acute rejection , observed after renal transplantation , has a 
 significant impact for long - term renal allograft survival .
even 
 though its incidence was reduced by new immunosuppressive drugs , 
 it remains a major problem after renal transplantation .
acute 
 rejection is a cell - mediated immune response that is initiated by 
 the recognition of cd t cells by the major 
 histocompatibility complex ( mhc ) class ii antigens on 
 antigen - presenting cells ( apcs ) of the graft 
 [ 14 ] . 

mhc class ii antigen activates cd t - lymphocytes , which 
 subsequently release cytokines .
released cytokines target vascular 
 endothelial cells , the first cells to be recognized by the host 's 
 immune system , and induce the expression of adhesion molecules and 
 chemokines implicated in t - lymphocyte adhesion and extravasation . 

initial transient adhesion of t - lymphocytes mediated principally 
 by selectins induce the rolling of the t - cells and their 
 subsequent activation and adhesion on the endothelium via other 
 adhesion molecules such as icam-1 and vcam-1 .
this adhesion stage 
 provides the necessary signal for full t - cell activation leading 
 to t - lymphocytes transendothelial migration .
endothelial cells forming the interface between donor and 
 recipients are the first donor cells to be recognized by the 
 host 's immune system .
endothelial cells coating the capillaries 
 that act as a barrier between the donor organ and recipient bears 
 the mhc class ii molecules .
they are highly responsive to 
 cytokines and express adhesion and other molecules implicated in 
 t - lymphocyte adhesion and extravasation . such endothelial control 
 in cell migration requires an effective intercellular adhesion , so 
 called cell junction , between the endothelial cells . 

adherent junctions mediate 
 the physical contacts between cells and anchor the actin 
 cytoskeleton .
cell - cell adhesion structures have been studied and 
 some membrane proteins   have been described such as pecam-1 
 or cd31 ; 
 51 and 
 21 integrins 
 , v - cadherin , and vascular endothelium cadherin ( ve - cadherin ) 
 [ 1012 ] .
the extravasation of lymphocytes in rejecting renal 
 allografts is thought to take place in activated peritubular 
 capillaries .
to date , the exact mechanisms involved in acute rejection after 
 solid organ transplantation are not completely understood . in a 
 previous work 
 
we have demonstrated that ve - cadherin is 
 under expressed in acute rejection in a murine heterotopic heart 
 transplantation model and in acute rejection following human heart 
 transplantation .
murine macroarrays results were validated in mice 
 and in humans by immunohistochemistry and quantitative 
 real - time - polymerase chain reaction ( q - pcr ) . in this study
we have 
 performed immunohistochemical staining of ve - cadherin in human 
 biopsies after renal transplantation .
we demonstrate that 
 ve - cadherin is also under expressed in acute rejection after renal 
 transplantation .
this study comprised transplant biopsy specimens from 15 patients 
 ( 66% of men with mean age of 47  15 years ) . all patients 
 received deceased donor kidney transplants .
after transplantation , 
 all patients received induction therapy with anti - lymphocyte 
 globulin or anti il-2 receptor and triple - therapy 
 immunossupression ( cyclosporine , steroids , and mycophenolate 
 mofetil ) .
details relating to important clinical parameters in 
 each group are given in table 1 .
needle kidney transplant biopsies were performed solely to define 
 the diagnosis and management of patients with acute deterioration 
 of allograft function in accordance with institutional guidelines . 

renal allograft tissues were obtained from transplant recipients 
 undergoing ultrasound guided biopsies from december 2002 to august 
 2003 .
biopsies were performed between 1 day and 16 years after 
 renal transplantation ( 453 , 4  1118 , 8 days ) .
specimens were 
 formalin fixed and paraffin embedded or snap frozen in liquid 
 nitrogen .
paraffin sections ( 4 m ) were stained with periodic acid 
 schiff ( pas ) and graded for acute rejection using the banff 
 criteria . 

fifteen kidney transplant biopsies showing no 
 rejection ( n = 5 ) , acute rejection grade ia ( n = 5 ) , or acute 
 rejection grade iia ( n = 5 ) were examined .
positive control was performed using 
 human spleen tissues that show a large number of endothelial 
 cells .
sections were incubated for 1 hour with a mouse antihuman 
 ve - cadherin monoclonal antibody ( 1:50 , chemicon international , 
 temecula , ca , usa ) followed by a goat antimouse immunoglobulin 
 ( dako , france ) , which was then revealed by the 
 streptavidin - biotin immunoperoxidase method .
sections were incubated for 1 hour with a mouse anti - human cd34 
 monoclonal antibody ( 1:50 , dako , france ) followed by an antimouse 
 biotin ( dako , france ) which was then revealed by the 
 streptavidin - biotin immunoperoxidase method .
sections were assessed by two independent observers ( ar , bm ) , 
 scored according to the number of ve - cadherin stained endothelial 
 cells in peritubular capillaries and the number of cd34 stained 
 cells .
sections were analyzed with a leica dmlb microscope ( leica 
 microsystems , germany ) directly coupled to a 3ccd colour camera 
 ( jvc , argenteuil , france ) .
quantification of ve - cadherin and cd34 
 staining was evaluated using image analysis software ( perfect 
 image , claravision orsay , france ) .
positive ve - cadherin and 
 cd34 labelled peritubular endothelial cells were counted in 10 
 high - power fields for each sample ( x630 magnification ) .
kruskal - wallis nonparametric test was used to compare clinical 
 characteristics between the 3 groups . in order to take into 
 account the variability between fields for each sample , variance 
 analysis for repeated measures
when this 
 global test was significant , fisher lsd post - hoc test was further 
 used when comparing between 2 groups .
the mean 
 time between transplantation and allograft biopsy was 
 431  1081 days ( range 14298 days ) .
there was no statistical 
 difference between donor age , donor and recipient sex or ischemic 
 time .
serum creatinine ( mean      sd ) was not statistically 
 different between groups .
all patients with histopathologic 
 diagnosis of acute rejection received steroid treatment after the 
 biopsy was obtained .
all human biopsies were constituted by adequate fragments 
 according to banff criteria 
  showing no acute rejection 
 ( n = 5 ) , grade ia acute rejection ( n = 5 ) , or grade iia acute 
 rejection ( n = 5 ) ( see figure 1 ) . in the no acute 
 rejection group the peritubular capillary endothelial cells have a 
 normal shape and an elongated cytoplasm . in the acute rejection 
 grade ia the lumen size is unchanged but populated with numerous 
 inflammatory cells adhering to swollen endothelial cells . in the 
 acute rejection iia ,
the lumen is dilated , some swollen 
 endothelial cells seemed to be detached from the basement membrane 
 and inflammatory cells have migrated from the lumen to the 
 interstitium .
ve - cadherin staining was differentially expressed between the 3 
 groups ( p = .02 ) . in no acute rejection group ve - cadherin
stained 
 cells represented 1.65  0.71% of the surface examined 
 compared to 1.62  0.34% in acute rejection grade ia and 
 0.75  0.31% in acute rejection grade iia ( see 
 figure 2 ) .
ve - cadherin expression was significantly 
 reduced in acute rejection grade iia when compared to no acute 
 rejection ( p = .01 ) and to acute rejection grade ia ( p = .02 ) . 

there was no difference in ve - cadherin expression between acute 
 rejection grade ia and no acute rejection group ( p = .7 ) . 

figure 3 shows ve - cadherin staining as a strong , thin , 
 linear staining on peritubular endothelial cells in renal biopsies 
 showing no rejection or acute rejection grade ia compared to acute 
 rejection grade iia .
cd34 staining confirmed the staining of 
 ve - cadherin on peritubular endothelial cells ( see 
 figure 3 ) .
the working hypothesis in this study is that the endothelium 
 lining the vessel wall of the donor kidney is first to be in 
 contact with host cells and is subjected to immunological 
 interactions .
these molecules can facilitate the 
 adhesion and transmigration of lymphocytes in the renal tissue 
 leading to damage and ultimately to graft failure .
we have 
 previously demonstrated by macroarrays , immunohistochemistry , and 
 q - pcr that ve - cadherin is under expressed in acute rejection 
 following murine and human heart transplantation . 
 in the 
 current investigation
, we bring evidence for the first time that 
 ve - cadherin is implicated also in acute rejection following renal 
 transplantation .
lymphocyte adhesion to the vascular lining accompanies the first 
 stages of the acute rejection reaction .
the ability of endothelial 
 cells to adhere lymphocytes is not a constant or a static 
 phenomenon .
several cytokines and adhesion molecules have shown to 
 increase lymphocyte binding to and penetration through endothelial 
 cells . in response to cytokines and to the 
 expression of endothelial cells adhesion molecules ,
binding to endothelium is 
 necessary , but not a sufficient request for the leukocyte to get 
 into tissue .
this stage is followed by a firm adhesion , and then 
 by a rapid transmigration of leukocytes through endothelial 
 intercellular junctions .
renkonen et al . 
 suggested and 
 confirmed the hypothesis that the peritubular capillary 
 endothelium is the site of entry of lymphocytes into rejecting 
 kidney allografts .
the authors showed that the increase in 
 lymphocyte binding to peritubular capillaries precede the peak of 
 leukocyte accumulation in the graft .
light and electron microscopy 
 revealed a marked activation of peritubular capillary endothelial 
 cells in allografts , whereas these alterations were less severe or 
 absent in syngenic controls and normal kidneys .
ve - cadherin is an endothelial - specific membrane protein , present 
 in adherent junctions of endothelial cells and responsible for the 
 endothelial cell - cell adhesion .
lampugnani 
 et al . 
 studied cultured endothelial cells monolayers by 
 immunofluorescence microscopy .
they described ve - cadherin as an 
 endothelial - specific membrane protein with a thin , sharp 
 continuous line highlighting the margins of each cell .
it was 
 present at the appositional surfaces of cultured cells only on 
 reaching confluency .
they observed that if the endothelial 
 permeability was increased its distribution was punctuated and 
 could be found only at intercellular contacts in a few areas . 

since then , various authors demonstrated the properties of 
 cell - cell adhesion of the ve - cadherin and the relationship of its 
 diminution with the increase of the endothelial permeability 
 [ 1720 ] . in our study , we found the same kind of staining described as a 
 thin , continuous line in the margins of renal peritubular human 
 endothelial cells .
cell adhesion molecules , mediating leukocyte 
 adhesion to endothelial cells , have been shown to affect gene 
 transcription in these cells .
previous studies 
 [ 1 , 11 , 
 12 ] 
 have demonstrated that through their adhesion , leukocyte could 
 transfer intracellular signals to endothelial cells in different 
 ways .
del maschio et al . 
 suggested that these 
 intracellular signals could induce endothelial 
 intercellular disorganisation .
they found that leukocyte adhesion 
 to endothelial cells was related to a reduced ve - cadherin 
 expression that could increase endothelial permeability .
an 
 alternative explanation for adherent junction 's disorganisation is 
 the release of lytic enzymes from the pmn that can be responsible 
 for ve - cadherin digestion and junction disassembly .
such 
 potential release of proteolytic enzymes , by activated 
 leukocytes , may lead to cleavage of the extracellular domain of 
 ve - cadherin , and an increase in the permeability in areas bearing 
 deposited leukocytes .
moreover , other authors 
 [ 23 , 24 ] 
 have 
 reported changes in cytosolic ca level in endothelial cells 
 during the adhesion of polymorphonuclear leukocyte .
the 
 polymorphonuclear leukocyte adherence could induce a series of 
 endothelial intracellular responses leading a detachment of 
 catenins from ve - cadherin .
it is conceivable that in acute 
 rejection after solid organ transplantation activated lymphocytes 
 adhering to vascular endothelial cells could affect the latter 
 cells by inducing the disappearance of ve - cadherin from 
 endothelial adherent junctions .
such action could result in a 
 significant increase in endothelial permeability due to the 
 disassembly of endothelial adherent junctions .
ve - cadherin was also investigated in other settings than acute 
 rejection after solid organ transplantation .
studied the immunohistochemical expression of 
 ve - cadherin in an animal model of renal ischemia .
the 
 authors observed that 24 hours after ischemia , the majority of the 
 renal microvasculature did not stain for ve - cadherin .
they 
 observed also that 72 hours after ischemia , ve - cadherin staining 
 in the renal microvasculature was similar to that observed under 
 physiological conditions .
studied the in 
 vitro effect of the uremic retention solute p - cresol observed in 
 chronic renal failure .
they observed an increase in endothelial 
 permeability associated with a decreased staining of junctional 
 ve - cadherin .
in summary , in this study we have demonstrated for the first time 
 a correlation between the expression of ve - cadherin present in 
 peritubular endothelial cells and acute allograft rejection after 
 human renal transplantation .
the under expression of ve - cadherin 
 in peritubular capillaries in acute rejection after kidney 
 transplantation could be responsible for the lymphocyte 
 transmigration into interstitial tissues leading to graft 
 dysfunction . preventing the down regulation of ve - cadherin could 
 conceivably prevent severe acute graft rejection .
 genes involved in acute rejection ( ar ) after organ transplantation remain to be further elucidated . in a previous work we have demonstrated the under - expression of ve - cadherin by endothelial cells ( ec ) in ar following murine and human heart transplantation . 
 serial sections from 15 human kidney banff - graded transplant biopsies were examined for the presence of ve - cadherin and cd34 staining by immunohistochemistry ( no ar ( n = 5 ) , ar grade ia ( n = 5 ) , or ar grade iia ( n = 5 ) ) . 
 
 quantification of peritubular ec staining were evaluated and results were expressed by the percentage of stained cells per surface analysed . 
 there was no difference in cd34 staining between the 3 groups . 
 ve - cadherin expression was significantly reduced in ar grade iia when compared to no ar ( p = .01 ) and to ar grade ia ( p = .02 ) . 
 this study demonstrates a reduced ve - cadherin expression by ec in ar after renal transplantation . 
 the down - regulation of ve - cadherin may strongly participate in human ar . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION