lupus nephritis ( ln ) in systemic lupus erythematosus ( sle ) is a major cause of morbidity and end - stage renal disease . ln develops in up to 60% of sle patients during the course of the disease . b cells are also considered to be involved in ln , particularly as a source of nephritogenic auto - antibodies . however , little is known about the role of b cells as part of the infiltrating cell population . this might be because b cells have classically been considered to exert long - range effects , mostly via activation in secondary lymphoid organs such as lymph nodes and the spleen , with subsequent proliferation and differentiation into antibody - producing plasma cells . studies have described the high prevalence of intrarenal b cells in immune - mediated diseases , including renal transplant rejection and glomerulonephritis [ 46 ] . local b - cell infiltrates could play a role in tissue injury such as tissue fibrosis , neolymphangiogenesis , and ectopic lymphomagenesis . recently , a contribution of b cells to the formation of lymphoid - like structures in renal tissue has been proposed . first examined b cells in ln patients and evaluated that most b cells displayed a mature non - antibody producing phenotype with antigen presenting ability . intrarenal b cells may be part of a local system that plays a pivotal role in the pathogenesis of ln . b - cell activating factor ( baff , also known as b - lymphocyte stimulator , blys ) belongs to the tumor necrosis factor ( tnf ) superfamily and can be produced by myeloid cells such as monocytes , macrophages , dendritic cells , and neutrophils . baff contributes to b - cell proliferation and differentiation , and it is important in immunoglobulin class switching . many researchers have demonstrated that high levels of baff may relax b - cell selection and contribute to autoantibody production , exacerbating proteinuria and renal inflammation in sle . tissue expression of baff has been found in germinal center b cells and/or plasma cells in lymph nodes of patients with sle . was the first to investigate baff in the kidneys of ln patients and found that baff was expressed in the interstitial inflammatory cell accumulation . we hypothesize that altered b - cell differentiation and tolerance induced by excess baff expression may be central to the pathogenesis of ln . in this study , renal b - cell infiltrates and baff expression were analyzed in human ln patients . by doing so , the relationship between b - cell infiltration and baff expression could potentially elucidate the mechanisms underlying ln . a prospective study of 62 patients who attended the department of rheumatology of renji hospital at the shanghai jiaotong university school of medicine was undertaken . all patients fulfilled the american college of rheumatology classification criteria for the diagnosis of sle . clinical evidence of ln was obtained in all cases , and pathologic findings from renal biopsy specimens confirmed the diagnosis . the following demographic , clinical , and serologic data were collected at the time of renal biopsy : sex ; age ; duration of sle and ln ; systemic lupus erythematosus disease activity index ( sledai ) ; levels of proteinuria , blood urea nitrogen , and serum creatinine ; and serum c3 , c4 , antinuclear antibodies ( ana ) , anti - sm , antiribonucleoprotein ( anti - rnp ) , antidouble - stranded dna ( anti - dsdna ) , and anti - nucleosome antibodies . the patients were informed of the purpose of the study and gave their consent for participation . plasma baff levels from 62 patients with ln and 40 healthy controls were detected using elisa kits according to the manufacturer 's instructions ( f00153 , westang , china ) . renal tissue obtained was fixed in 10% neutral buffered formalin , dehydrated gradually , and embedded in paraffin . paraffin - embedded tissue sections were stained with hematoxylin and eosin , periodic acid - schiff , masson 's trichrome , and periodic acid - silver methenamine . small portions of fresh renal tissue were snap - frozen , and 4  m cryostat - cut sections were incubated with fluorescein isothiocyanate- ( fitc- ) conjugated rabbit antisera against human igg , iga , igm , c1q , and c3 ( dako , denmark ) , and direct immunofluorescence of these sections was examined . the biopsy specimens were classified using the international society of nephrology / renal pathology society ( isn / rps ) 2003 classification of ln . renal tissue injury was evaluated on the basis of activity and chronicity indices according to methods reported by austin et al . . the activity index was calculated as the sum of the scores ( on a scale of 13 ) of endocapillary proliferation , karyorrhexis , fibrinoid necrosis ( the score was multiplied by 2 ) , cellular crescents ( the score was multiplied by 2 ) , hyaline deposits , leukocyte exudation , and interstitial inflammation . the chronicity index was the sum of the scores ( on a scale of 13 ) for glomerular sclerosis , fibrous crescents , tubular atrophy , and interstitial fibrosis . as previously described , we used serial sections for each patient . cd20 monoclonal antibody ( l26 , dako ; glostrup , denmark ) and baff monoclonal antibody ( alx-804 - 131-c100 , enzo life sciences international inc . ) were used as immunohistochemical staining to identify infiltrating b cells and baff expression . briefly , the paraffin - embedded tissue sections were placed on positively charged slides and incubated in a stove at 60c for one hour . the sections were deparaffinized and rehydrated through a series of washes with xylene and graded alcohol . antigen retrieval was performed by flooding the slides with edta - tris buffer ( ph 9.6 ) and heating in a microwave at 1,000  w for 10 minutes . primary rat monoclonal anti - human baff antibody was applied to the slides at a dilution of 1  :  50 in 1% bovine serum albumin / phosphate buffered saline ( bsa / pbs ) , and the slides were subsequently incubated overnight at room temperature . the slides were then incubated with a secondary goat anti - rat igm antibody ( alx-211 - 055-r050 , enzo life sciences international inc . ) for 30 minutes . these sections were then washed with pbs ( ph 7.4 ) between each step ( 3 times , 5 minutes each time ) . finally , the sections were counterstained with mayer 's hematoxylin , air - dried , cleared in xylene , and coverslipped . positive controls were performed on sections of healthy human lymph node with a nonimmune rabbit serum . the slides were counterstained with haematoxylin and observed under a leica light microscope . in light microscopy positive parts show brown . results of the renal biopsies from the 62 patients with ln were classified according to the organizational stage of b - cell infiltrates . three distinct categories could be distinguished : no infiltrate of intrarenal cd20-positive b cells was graded as 0 ( group 0 ) ; a scattered pattern of intrarenal cd20-positive b cells was graded as 1 ( group 1 ) ; and nodular aggregates consisting of cd20-positive b cells were graded as 2 ( group 2 ) . the results of intrarenal staining for baff were scored as 0 , 1 , or 2 , where 0 represented absence of baff staining , 1 represented weak baff staining , and 2 represented moderate or strong staining . typical examples of baff expression in consecutive tissue samples are shown in figures 2(a)2(d ) . all the aforementioned biopsy specimens were scored by a renal pathologist who had no prior knowledge of the clinical and laboratory findings of the patients . categorical variables were compared using fisher 's exact test or the chi - square test . differences between the median values of defined patient groups were compared using the nonparametric mann - whitney u test . in this study , we prospectively examined 62 ln patients ( 58 women and 4 men ) with a mean age  sd of 31  11 years . depending on the presence or not of b - cell infiltrates in renal biopsies , the patients were divided into two groups , that is , those with b - cell infiltration ( the ln - b group ) and those without ( ln - non - b group ) . there were 35 ( 56.5% ) patients in the ln - b group ( 32 women and 3 men ) and 27 ( 43.5% ) patients in the ln - non - b group ( 26 women and 1 man ) . the mean age  sd of the two groups was 33  11 and 29  11 years , respectively . no significant difference was seen between the groups in terms of age or gender ( p > 0.05 for all ) . proteinuria ( g/24 hours ) , blood urea nitrogen , and serum creatinine levels were all significantly greater in the ln - b cell group than in the ln - non - b cell group ( p < 0.05 for all ) . however , the duration of sle or ln , sledai , as well as the level of c3/c4 , was not statistically different between the two groups . in addition , we failed to find any association between the presence of intrarenal b - cell infiltration and that of anti - dsdna antibodies ( p > 0.05 ) ( table 1 ) . immunohistochemical staining , 27 ( 43.5% ) patients were classified grade 0 , 23 ( 37.1% ) grade 1 , and 12 ( 19.4% ) grade 2 . we found that the level of proteinuria ( g/24 hours ) in grade 0 was lower than other grades ( p < 0.05 for all ) , and there was no statistically difference between grade 1 and 2 ( p > 0.05 for all ) . the same results in serum creatinine and blood urea nitrogen level ( figure 3 ) . all biopsy samples from the patients were stained for cd20 as a pan b - cell marker . b cells were mainly distributed in the renal tubules , the renal interstitial vessels , and in the interstitium , but rarely in glomerular tissue ( figure 1 ) .   the distribution of the isn / rps classification of the patients was as follows : 3 were class i , 2 were class ii , 4 were class iii , 22 were class iv , 14 were class v , 11 were class ( iii + v ) , and 6 were class ( iv + v ) . in the ln - b - cell group , none of the biopsies were class i or class ii , 3 were class iii , 17 were class iv , 6 were class v , 4 were class iii + v , and 5 were class iv + v. when compared with the ln - non - b - cell group , the ln - b - cell group was more likely to be associated with class iv ln ( p = 0.0142 ) ( table 2 ) . within the three different groups of ln patients , we found that the activity and chronicity indices of grade 0 were significantly lower than those of the other grades ( p < 0.05 for all ) , but there was no significant difference between grade 1 and grade 2 ( p > 0.05 for all ) ( figure 4 ) .   for baff expression in tissue samples from patients with ln , there were 32 ( 52% ) patients with grade 0 , 16 ( 25% ) patients with grade 1 , and 14 ( 23% ) patients with grade 2 . similar to the b - cell group , urine protein ( g/24 hour ) , blood urea nitrogen , and serum creatinine levels were all higher in the baff expression group ( p < 0.05 for all ) , with no statistically significant difference between grade 1 and grade 2 ( p > 0.05 for all ) . baff protein was mainly expressed in infiltrating inflammatory cells of the renal interstitial tissue , with very few seen in glomerular tissue ( figure 2 ) . enhanced baff protein was most expressed in class iv ln ( 50% ) and rare in class v ln ( 13% ) ( p = 0.0330 and p = 0.0227 , resp . ) . the baff expression group demonstrated higher activity and chronicity indices than the non - baff expression group ( p < 0.05 for all ) , with no significant difference between grades 1 and 2 ( p > 0.05 for all ) . the expression of baff was strongly associated with the amount of b - cell infiltration in the interstitium ( r = 0.6226 , p < 0.0001 ) ( table 3 ) . there was evidence of strong expression of baff in the area of b - cell infiltrates in kidney samples from ln patients , although they did not completely overlap ( figure 5 ) . there was no significant correlation between plasma baff levels and the degree of expression of baff protein in patients with ln ( data not shown ) . there was no statistically significant difference between the b - cell infiltrating group and the non - b - cell infiltrating group ( p > 0.05 , data not shown ) . lupus nephritis is one of the most frequent and serious complications for patients with sle and has a profound effect on both morbidity and mortality . dysfunction of the b cells , an important component of adaptive immunity , is thought to be important in the pathogenesis of sle / ln . the production of pathogenic antibody has been traditionally viewed as the principle contribution of b cells to the pathogenesis of immune - mediated glomerulonephritis . however , it is increasingly appreciated that b cells have a much broader role in such diseases , functioning as antigen - presenting cells , regulators of t cells , macrophages , and dendritic cells , as well as being involved in the formation of local lymphatic expansion .   in this study , we prospectively analyzed 62 renal biopsies , which were then classified into different groups . as we have demonstrated , b cells are almost exclusively detectable in the tubulointerstitial compartment and rarely in the glomerular tuft . there was a significant correlation between intrarenal b cells and the degree of renal function . in our study , we found that proteinuria ( g/24 hour ) , blood urea nitrogen , and serum creatinine levels were all significantly greater in the ln - b - cell group than in the ln - non - b - cell group . furthermore , intrarenal b cells were more likely to be associated with class iv ln , and the activity and chronicity indices were also significantly higher in the ln - b - cell group compared with the ln - non - b - cell group . therefore , it can be speculated that the formation of interstitial b - cell aggregates is a common response in ln and plays a pivotal role in renal injury . b cells are recruited to most chronically inflamed tissues , and resembling secondary lymphoid tissue has been found in several forms of inflammatory kidney diseases , including renal transplant rejection and lupus nephritis . their pathophysiological significance remains ill defined . recent studies on ln patients have revealed that intrarenal b cells can form local lymphoid tissue , called tertiary lymphoid neogenesis , which display a mature non - antibody producing phenotype with antigen presenting ability . recently , chang et al . reported that in some lupus renal biopsies there were germinal center- ( gc- ) like structures which were functional , as they were associated with in situ b - cell clonal expansion and somatic hypermutation . in b - cell immunohistochemical staining , we found that 23 ( 37.1% ) were of the scattered pattern ( grade 1 ) , and 12 ( 19.4% ) demonstrated a nodular aggregates pattern ( grade 2 ) , which resembled germinal center- ( gc- ) like structures , suggesting ectopic germinal center formation , as mentioned previously . these different results may possibly reflect heterogeneity in ln pathogenesis and also emphasize the need for further research into the role of intrarenal b cells in ln . as a new member of the tnf superfamily of ligands , b - cell activating factor ( baff ) was first discovered in 1999 and on binding to its receptors ( baff - r ) exerts strong stimulation on b cells , promoting b - cell survival and maturation . april ( a proliferation - inducing ligand ) considerable similarities with baff , april is more important for plasma cells survival . but in the phenotype of april transgenic ( tg ) and april knock - out mice is much less dramatic than that of the baff tg and knock - out mice ; thus we focus on baff . increased serum and/or plasma levels of baff have been documented in several human autoimmune diseases such as sle , rheumatoid arthritis , and sjogren 's syndrome , indicating the involvement of baff and the overactivation of b cells in the pathogenesis of these autoimmune diseases [ 21 , 22 ] . demonstrated baff expression on infiltrating cells in salivary gland tissue from patients with sjogren 's syndrome , suggesting the importance of baff signaling in disease pathogenesis . in patients with rheumatoid arthritis , concentrations of baff in synovial membrane were much higher than in corresponding health control samples . however , detailed data on local renal baff expression in human lupus nephritis is scarce . neusser et al .   first demonstrated that baff and baff - r were expressed in interstitial inflammatory cell accumulates in ln patients . taci expression tended to increase in sle patients , especially in ln patients , which indicating that taci may be a suppressive receptor of baff . over - expression of taci in patients with ln suggested that taci may be involved in the pathogenesis of nephritis . in our study , we also found that baff was expressed in renal interstitial tissue , most infiltrated in class iv ln and has elevated proteinuria ( g/24  h ) , blood urea nitrogen , and serum creatinine levels . our experiment showed an abundant expression of baff in the area of b - cell infiltrates in renal interstitial tissue . the expression of baff was strongly associated with the amount of b - cell infiltration . since the baff expression area did not exactly overlap with the b - cell infiltrates area , we can not be certain that functional baff is released by activated b cells . in order to demonstrate that baff expression could be induced by infiltrating inflammatory cells , such as monocytes , macrophages , dendritic cells , and neutrophils , further research using confocal microscopy clinical trials on anti - baff have been investigated in sle , and the he blockade of the baff system appears to demonstrate some promising results . hence , baff - targeting therapy , by using therapeutic agents that block baff activity , is a promising area currently undergoing clinical trials , for the treatment of b - cell - related autoimmune diseases , particularly sle . understanding the renal expression patterns of b cells and their effectors could help physicians in their choice of appropriate targeting reagents for different patients , but further research is needed in this area . in conclusion , this study discussed correlations between clinical parameters , and the presence of different intrarenal b cell distributes in a large number of ln patients . as baff expression was strongly associated with b - cell infiltration , we hypothesize that altered b - cell differentiation and tolerance induced by excess baff may be central to the pathogenesis of ln pathogenesis further research is needed to shed more light on the function of intrarenal b cells .
introduction . 
 dysfunction of the b lymphocyte is considered to be involved in the pathogenesis of lupus nephritis ( ln ) . 
 intrarenal b cells have been found in several forms of inflammatory kidney disease . 
 b - cell activating factor ( baff ) regulates b lymphocyte proliferation and survival , and contributes to human autoimmune disease . 
 their role in renal inflammation is not well defined . 
 methods . clinical parameters and renal biopsies from 62  ln patients were prospectively analyzed . 
 we performed standard immunohistochemistry on serial paraffin tissue sections using monoclonal antibodies to cd20 and baff to investigate the characteristics and significance of locally infiltrating b cells and local baff expression in patients with ln . 
 results . 
 intrarenal b cells and/or baff were mainly distributed in the renal interstitium . compared to the ln - non - b - cell / 
 baff expression group , proteinuria ( g/24 hour ) , blood urea nitrogen , serum creatinine levels , ln renal activity , and chronicity indices , were all significantly greater in the ln - b - cell / baff expression groups . 
 the expression of baff was strongly associated with the quantity of b - cell infiltrate in the interstitium . conclusion . 
 as baff expression was strongly associated with b - cell infiltration , we hypothesize that altered b - cell differentiation and tolerance induced by excess baff may be central to the pathogenesis of ln .