atherosclerosis is a dynamic inflammatory disease [ 1 , 2 ] in which autoantigenes , such as oxidized low - density protein ( oxldl ) , play an essential role . dendritic cells ( dcs ) are key regulatory antigen - presenting cells ( apcs ) that induce inflammatory processes . dcs can be detected in the vascular inflammatory environment , from endothelial dysfunction to consecutive plaque formation and rupture [ 35 ] . dcs are the most potent apcs and are highly specialized to prime naive t - cells . interestingly , mature dc - specific markers , for example , cd83 , accumulate during plaque progression . it has been shown previously that the presence of cd83 is more than two fold , higher in symptomatic compared to asymptomatic patients . dcs , after entering the vascular tissue , screen the environment for potential antibodies . after processing the antigens , a maturation process is initiated and immunomodulatory receptors , such as the cd83-receptor ( important for t - cell - stimulation ) , are upregulated . in a previous study , we found a cd83 upregulation on dcs by proatherosclerotic stimuli like oxldl and asymmetric dimethylarginine ( adma ) . beside cd83 , dc induces ccr7 expression during maturation , which acts as a receptor for constitutively expressed ccl21 and ccl19 [ 10 , 11 ] . in analogy to dc accumulation and maturation , activated ccr7 expressing t - cells are trapped especially in the plaque shoulders [ 12 , 13 ] where mature dcs are forming clusters with t cells . several studies have demonstrated the important link between dc / t - cell recruitment and ccr7 expression [ 1214 ] . the increase in ccr7 receptors induces homing of mature dcs and t - cells to the lymph nodes through ccl21/ccl19 expressing lymph vessels [ 2 , 11 , 12 ] . high plasma concentrations of oxldl and their appearance in atherosclerotic lesions are of utmost importance in the pathogenesis of atherosclerosis . the present study focuses on the influence of oxldl on the dc - related chemokine receptors ccr7 , ccl19 , and ccl21 . we characterized the dc - specific chemokine - ligand expression in human atherosclerotic carotid artery plaques . furthermore , we investigated the impact of oxldl on the dc receptor ccr7 expression and its ligands ccl-21 on human microvascular endothelial cells ( hmecs ) . all investigations were approved by the institutional review board and the ethics committee of the ludwig - maximilians university of munich . the investigation conforms to the principles outlined in the declaration of helsinki . between september 2006 and may 2008 , carotid endarterectomy ( cea ) the indications for cea were based on carotid duplex sonography : stenosis of the internal carotid artery of more than 70% for symptomatic patients and for comparison , we collected 10 blood samples from healthy age - matched men without clinically manifested atherosclerosis and 14 pieces of aortic tissue with no visible atherosclerotic lesions . 50  pg / tube rna ( pbmcs ) was used . first - strand cdna synthesis and pcr the two - step quantitative real - time pcr ( rt - pcr ) system was applied according to the manufacturers ' instructions . the quantitative real - time pcr system provides optimal performance with sybr green primers ( qiagen : hilden , germany ) . rt - pcr was performed in the abi prismtm 7700 system ( applied biosystems , germany ) . data analysis was performed using the delta - delta - ct method as described previously . primer sequences for the amplified fragments were gapdh : 5-cgg agt caa cgg att tgg tcg tat-3/5-agc ctt ctc cat ggt ggt gaa gac-3 ; ccr7 : 5-tgg agg cct tta tca cca tc-3/5-tgt agg gca gct gga aga ct-3 ; ccl19 : 5-ctg tga ccc aga aac cca tc-3/5-gct tca tct tgg ctg agg tc-3 ; ccl21 : 5-ccc agc tat cct gtt ctt gc-3/5-tca gtc ctc ttg cag cct tt-3 ; cd4 : 5-agg aag tga acc tgg tgg tg-3/5-ctc agc aga cac tgc cac at-3.   frozen sections ( 10  m ) were prepared from human aorta and plaque material of the internal carotid artery . after fixation in methanol / ethanolic acid for 1.5  min at 20c , the sections were washed immediately 3 times for 3  min in pbs . unspecific binding was blocked with a 2% bsa/0.2% tween 20 solution for 1  h and followed by 3 washing steps in pbs . to visualize nucleoli we used dapi ( 4 , 6diamidino-2phenylindole , merck kgaa , germany ) in a final concentration of 1  g / ml . after another washing step the sections were incubated with a fluorescent labelled anti - human ccl21/6ckine antibody ( 5  g / ml , r&d systems , inc . , germany ) . to determine the localization of the ccl21 expressing structure ( ccl21/6ckine antibody 5  g / ml , r&d systems , inc . , germany ) , the sections were also incubated with a cy3-labeled anti - pecam-1 antibody ( 5  g / ml , abd serotec , germany ) , which demarcates the endothelia cells in the vessel wall . immunofluorescence analysis of ccl21 expression was investigated by confocal microscopy ( lsm 510 meta , zeiss , plan - neofluar 40x/1.30 oil objective , germany ) and quantified by lsm image browser 4.2 ( zeiss , germany ) . the sections were incubated with an fitc - labelled anti - human ccr7 antibody ( 1  :  100 concentration , r&d systems , inc . , germany ) for 1  h , followed by 3 washing steps in pbs . the sections were incubated with an atto594-labeled anti - cd4 antibody ( 10  g / ml , abd serotec , germany ) or atto594-labelled anti - cd83 ( 10  g / ml , abd serotec , germany ) for 1  h , which represents the t - lymphocytes ( cd4 ) or mature dc ( cd83 ) . the immunofluorescence analysis was investigated by confocal microscopy ( lsm 510 meta , zeiss , plan - neofluar 40x/1.30 oil objective , germany ) . oxldl levels were examined by using a cytokine - specific elisa kit according to the manufacturers ' instructions ( immundiagnostik , bensheim , germany ) . before analysis , samples were treated as described above . in brief , after washing with wash buffer several times , standard samples and controls were added and incubated for 4 hours at room temperature on a horizontal mixer . thereafter , a washing conjugate was added and incubated for 1  h as described before . the substrate was added and incubated for 25 minutes in the dark . stop - solution was added and immediately analyzed by an elisa - reader at 450  nm . detection of ccl21 in serum was also performed by a quantitative sandwich enzyme immunoassay technique , according to the manufacturers ' instructions ( r&d systems , germany ) . mononuclear cells were isolated from 100  ml of peripheral blood of a healthy human donor by a ficoll density gradient according to the protocol by boyum . the purity of the monocyte culture was enhanced up to 97% by adhesion on -globulin coated plates . dcs were obtained from the monocyte culture according to the modified protocol by romani et al .   as described in detail in our recent publication [ 9 , 17 ] . all experiments were performed with human microvascular endothelial cells ( hmecs-1 ) , generously provided by the center for disease control and prevention and the national center for infectious disease ( atlanta , usa ) . cells were cultured in mcdb-131 ( without phenol red ; cc pro , neustadt / w . , germany ) supplemented with 10% fetal calf serum ( fcs ; paa , pasching , austria ) , 2  mm l - glutamine ( biochrom ag , berlin , germany ) , 1  g / ml hydrocortisone , and 10  ng / ml epidermal growth factor ( sigma , taufkirchen , germany ) . the cells were used at least 10 days after thawing and for no more than 20 passages . ldl ( density = 1.019 to 1.063  g / ml ) was isolated from human plasma of normolipidemic healthy volunteers by sequential ultracentrifugation as described and stored in pbs containing 2  mmol / l edta . shortly before oxidation , the edta was removed from ldl by passing the lipoprotein through a pd 10 column ( pharmacia , austria ) . this reflects oxldl concentrations in the plaque and has been used in previous studies [ 5 , 22 , 23 ] . in addition , several other studies have shown that an oxldl concentration of ( 10  g / ml ) is not toxic to the cultured cells [ 9 , 24 , 25 ]   for isolation of mrna from hmecs-1 and dcs , the total rna isolation rnaeasy mini kit from qiagen ( hilden , germany ) was used according to the instructions provided by the manufacturer . 50  pg / tube rna ( pbmc ) was used . first - strand cdna synthesis and pcr the two - step quantitative rt - pcr system was applied according to the manufacturers ' instructions and as described above . antibodies were matched to iso - type - controls ( mouse-2a-(fitc)/-1(pe)-fastimmune ; bd ; usa ) . to verify the purity of our dc - culture , we used cd3 ( bd , usa ) and cd20 ( bd , usa ) to rule out a t - cell and b - cell contamination ( < 5% ) . dcs were characterized by low cd14 expression ( bd , usa ) and high expression of cd80 ( bd , usa ) , cd86 ( bd , usa ) , and hla - dr ( bd , usa ) , as described previously . at day five , we incubated the dcs with oxldl ( 10  g / ml ) for 24 hours . expression analysis of the chemokine receptor ccr7 ( r&d , usa ) and cd83 ( biolegend , usa ) on human dcs were performed using flow - cytometer analysis [ 16 , 17 ] . hmecs-1 were cultivated on chamber slides and stimulated with 10  g / ml oxldl for 48  h. the monoclonal antibody for ccl21/6ckine ( r&d , usa ) was labeled with alexa 488 using the alexa fluor protein labeling kit ( invitrogen , life technologies , usa ) according to the manufacturers ' instructions . hmecs-1 were incubated with the labeled antibody ( concentration 1  :  20 ) for 30 minutes . thereafter , vybrant did ( invitrogen , life technologies , usa ) was used as a marker of the plasma membrane . ccl21-labeled hmecs-1 were investigated by confocal microscopy ( zeiss lsm 510 meta , plan - apochromat 63x/1.40 oil objective ) and quantified by lsm image browser 4.2 ( zeiss , germany ) . the kolmogorov - smirnov test was used to determine whether or not the data was normally distributed . if the data was normally distributed , the unpaired t - test was used to compare the two groups . differences between means were considered significant with p < 0.05 and highly significant with p < 0.01 . all in - vitro experiments were repeated at least eight times with different cells and lipoprotein preparations . carotid plaque tissue and blood samples were collected from 47 patients undergoing cea , of which 77% were male and , on average ,  70  8  years of age . serum for the pbmcs in the control group was collected from 10 healthy age - matched patients . none had cerebral ischemic complications or clinical manifest atherosclerosis . concerning the risk - profile , 20% had insulin - dependent diabetes mellitus , 80% hypertension , and 10% were smokers . concerning the medication , 10% took thrombocyte aggregation inhibitors , 80% statins , and 60% had a -blocker ( table 1 ) . aortic tissue in the control group was collected from 14 patients who underwent valve replacement and had no sign of aortic atherosclerosis . 71% were of male gender with an average age of  64  5  years . concerning the risk profile , 28% had insulin - dependent diabetes mellitus , 85% hypertension , and 35% were smokers . with respect to medication , 43% took thrombocyte aggregation inhibitors , 78% statins , and 43% had a -blocker . in line with previous investigations , we detected that the median cd83 mrna levels in plaque tissue from cea - group were nearly twice as high as in healthy aortic tissue ( p < 0.01 ) , demonstrating the augmented presence of mature dcs in the atherosclerotic tissue [ 7 , 27 , 28 ] . in a subgroup analysis , we further found that cd83 levels in men were significantly higher ( + 120% ; p < 0.05 ) compared to women . cd4 mrna levels were even  23  11  fold higher in healthy aortic tissue ( p < 0.01 ; data not shown ) . ccr7 , normally coexpressed on mature dcs and t - cells , was 81% lower in the plaque compared to compared to healthy aortic tissue ( p < 0.01 ) without any gender specific differences . further analysis of ccl19 revealed that its transcripts were downregulated by 99% ( p < 0.01 ) . ccl21 was found 90% lower in the plaque ( p < 0.01 ) compared to healthy aortic tissue ( figure 1 ) . furthermore ccl21 was also found lower in the symptomatic patients ( 58% ; p < 0.05 ) compared to asymptomatic patients from the cea group .   to confirm the pcr results on the protein level , we performed a semiquantitative immunofluorescence analysis on histological slides of human aortic and plaque tissue . to analyze the location of ccl21 expressing cells this revealed a colocalization of the ccl21 and the vascular wall of the vasa vasorum ( figure 2(a ) ) . in every single healthy aortic tissue slide ( n = 6 ) , we could detect higher ccl21 expression via an increased fluorescence signal signal from the binding fluorescent antibody than in the plaque tissue ( n = 15 ) . an example is shown ( n = 15 ; in figure 2(b ) ) . to further investigate differences of the ccr7 level on t - lymphocytes ( cd4 positive ) and mature dc ( cd83 positive ) in human tissue , , we could detect a decreased signal of ccr7 on cd4-positive cells within the plaque tissue ( figure 2(c ) ; lower panel ) compared with control aortic tissue ( figure 2(c ) ; upper panel ) . also , cd83-expressing cells within the plaque show a decreased ccr7 signal ( figure 2(d ) ; lower panel ) compared to the aortic tissue which correlates with the pcr results . for oxldl , we found a 52.1% ( p < 0.05 ) higher concentration in the plaque group compared to the control group ( table 1 ) . oxldl serum concentrations showed no correlation with ccl21/ccl19 or ccr7 expressions in plaques ( table 1 ) . < 0.01 ) higher serum concentration in the cea group compared to the controls ( table 1 ) . after stimulation with oxldl , we found a reduction of the mrna levels of ccr7 ( dcs ) by 30% using rt - pcr ( n = 8 ; p < 0.05 ) . this correlated with a reduction in protein expression by 46% ( n = 8 ; 25.7  1.06% versus control 47.6  19.3% positive cells ; p < 0.05 ; figure 3 ) . in contrast , protein expression of cd83 was significantly upregulated by 10  g / ml oxldl , as shown in figure 3 ( 60% , 46.9 p < 0.05 ; figure 2 ) by 10  g / ml oxldl ( figure 3 ) . for hmecs-1 , stimulation with oxldl led to a significant reduction of ccl21 mrna expression ( 50% ; ct = 0.5 ; p < 0.05 , figure 4(a ) ) . these results correspond with the results of the immunofluorescence analysis , where we found a 24% down - regulation of ccl21 receptor expression ( n = 9 ; 34.00  8.53 versus control 44.54  6.23 intensity / mm ; p < 0.05 ; figures 4(b ) and 4(c ) . oxldl had no significant impact on protein or mrna expression of ccl19 ( figure 4(a ) ) . the main findings obtained from our study are ( 1 ) the dc - chemokine receptor ccr7 and its ligands ccl21/ccl19 are significantly downregulated in atherosclerotic plaques ; ( 2 ) circulating ccl21 levels are upregulated in serum from atherosclerotic patients ; ( 3 ) oxldl impairs ccr7 expression in dcs and ccl21 expression in microvascular ecs . our data support the concept that modulation of chemokine receptors ( mediated , e.g. , by oxldl ) in the plaque may trigger retention of dcs , thereby impeding the vascular innate and adaptive immunity [ 29 , 30 ] . accordingly , angeli et al . found that oxldl and other lipid mediators are jointly responsible for trapping of dcs in the vascular wall . in our in vivo study , however , ccr7 and ccl21/ccl19 were downregulated in atherosclerotic plaques compared to nonatherosclerotic aortic tissue . this discrepancy may be explained by the fact that oxldl is unstable in serum but accumulates in the subintimal space over time . hereby , it reaches much 70 fold higher subintimal concentrations as compared to serum levels . alongside , ccl21 was downregulated on hmecs-1 after incubation with oxldl , in a concentration which predominates in atherosclerotic plaques . previously demonstrated that phases of hyperlipidemia severely reduce ccr7 mrna and the corresponding protein levels in an apoe - mice model . ccr7 expression was found to be essential for the migration of dcs from atherosclerotic plaque . in atherosclerotic plaques of apoe - mice , this process was accompanied by an upregulation of ccr7 itself . under a high - dose statin therapy , damas et al . found a significant decrease in ccl19 and ccl21 levels in serum . our data support this observation , showing reduced ccl21 serum protein expression in the control group , characterized by low oxldl levels . our results suggest that dc maturation is triggered by increased oxldl deposits in the subintimal space . at the same time dcs , and even t - cells do not seem to express enough ccr7 receptors in order to migrate . as a consequence , oxldl , dcs and t - cells appear to accumulate , thereby enhancing plaque inflammation processes and possibly plaque rupture . these results complement the former findings that oxldl increased the adhesion and promotes the maturation and differentiation of dcs from monocytes [ 9 , 26 ] . furthermore , oxldl supports the building of foam - cell accumulations and also directly induces chemotaxis of immune cells like t - cells via upregulation of endothelial adhesion molecules . keeping in mind that dcs are responsible for priming naive t - cells to oxldl - specific t - cells [ 9 , 33 ] , the interaction of oxldl and dcs must be taken into account as a key factor for the induction and progression of atherosclerosis . oxldl not only seems to have an influence on the expression of ccr7 , but also to induce downregulation of ccl21 on ecs in vitro as well as decreased expression of ccl21 and ccl19 in the plaque . comparing ccl19 with ccl21 , the latter appears to be affected more directly by local plaque homing factors . in plaque versus normal aortic tissue , we found a much higher response to ccl21 on ecs as opposed to ccl19 . typically , the expression of ccl21 on ecs is higher than ccl19 , since ccl21 has a c - terminus indicating a strong affinity to glycosaminoglycan . this is crucial for effective presentation of ccl21 on ecs [ 11 , 34 ] . in contrast to our study , other investigators demonstrated an upregulation of ccl21/ccl19 in carotid plaques . in previous studies , 24 coronary or renal vessels from autopsy material were taken as controls . to overcome these limitations , we used 14 age - matched healthy aortic tissue samples taken from valve operations serving as controls , which were immediately frozen after their operative preparation , similar to the procedures of carotid tissue preparation . autolysis and coincidence caused by the small number of controls were therefore ruled out in our experimental setting . a further aspect which points against the renal vessels as controls is the fact , that the vascular - associated lymphoid tissue ( including dc and t - cells ) has been characterized in human large arteries , including the aorta , carotid , and coronary arteries [ 35 , 36 ] but not in renal vessels . in summary , our results suggest that alterations in the vascular chemokine profile may be responsible for accumulation of mature dcs in plaque , which potentially enhance the risk for plaque rupture . it may be argued that inhibiting dc migration leads to a less robust immune response . indeed , altered priming can be expected in the presence of impaired dc migration . however , if migration is blocked and maturation enabled , the consequence of trapping mature dcs in the direct atherogenic vicinity may induce plaque formation and progression or even rupture . suppression of dc migration and maturation may , therefore , prove a promising future therapeutic target to prevent plaque instability and rupture .