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 .
 
 introduction . 
 dendritic cells ( dcs ) and oxldl play an important role in the atherosclerotic process with dcs accumulating in the plaques during plaque progression . 
 our aim was to investigate the role of oxldl in the modulation of the dc homing - receptor ccr7 and endothelial - ligand ccl21 . 
 methods and results . 
 the expression of the dc homing - receptor ccr7 and its endothelial - ligand ccl21 was examined on atherosclerotic carotic plaques of 47 patients via qrt - pcr and immunofluorescence . in vitro 
 , we studied the expression of ccr7 on dcs and ccl21 on human microvascular endothelial cells ( hmecs ) in response to oxldl . 
 ccl21- and ccr7-mrna levels were significantly downregulated in atherosclerotic plaques versus non - atherosclerotic controls [ 90% for ccl21 and 81% for ccr7 
 ( p < 0.01 ) ] . in vitro , 
 oxldl reduced ccr7 mrna levels on dcs by 30% and protein levels by 46% . 
 furthermore , mrna expression of ccl21 was significantly reduced by 50% ( p < 0.05 ) and protein expression by 24% in hmecs by oxldl ( p < 0.05 ) . 
 conclusions . 
 the accumulation of dcs in atherosclerotic plaques appears to be related to a downregulation of chemokines and their ligands , which are known to regulate dc migration . 
 oxldl induces an in vitro downregulation of ccr7 and ccl21 , which may play a role in the reduction of dc migration from the plaques . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Discussion
5. Conclusions