rheumatoid arthritis ( ra ) is a systemic autoimmune disease characterized by synovitis and joint destruction , leading to severe deformity and disability without proper therapy . previous studies showed that ra patients with anticyclic citrullinated peptide antibodies ( anti - ccp ab ) had more swollen joints and more severe radiological destruction than those without anti - ccp ab [ 1 , 2 ] . another study indicated that ra patients with high titer of anticollagen type ii antibody may have a distinct clinical phenotype with significantly elevated c - reactive protein ( crp ) , erythrocyte sedimentation rate ( esr ) , tnf- , il1- , and il-8 at baseline . recently , much attention has been paid to igg4 since the recognition of igg4-related disease ( igg4-rd ) , a new emerging disease entity . igg4-rd is a systemic disease characterized by swelling or masses in the involved organs , elevated serum igg4 ( sigg4 ) , and marked igg4-positive plasma cells infiltration and fibrosis . elevation of serum and histological igg4 separates mikulicz 's disease   from sjgren syndrome and type 1 autoimmune pancreatitis   from autoimmune pancreatitis . it was reported that sigg4 elevated in ra patients compared to healthy control [ 7 , 8 ] . however here we explored the correlation of igg4 with clinical manifestations and therapeutic response in ra . one hundred and thirty - six consecutive ra patients who fulfilled 1987 acr revised classification criteria for ra or the 2010 acr / eular classification criteria for early ra were recruited from april 2010 to january 2013 at sun yat - sen memorial hospital , sun yat - sen university , guangzhou . patients with allergic disorders , pemphigus , parasite infestations , castleman 's disease , churg - strauss syndrome , or igg4-rd were excluded . the study was approved by the medical ethics committee of sun yat - sen memorial hospital and all patients signed informed consent . all patients were followed up at regular interval . demographic characteristics , ra clinical assessments , and therapeutic regimens were collected at baseline and 1st , 3rd , and 6th months . ra clinical assessments include the core set of disease activity measures for ra recommended by acr   and measurement of three autoantibodies : rheumatoid factor ( rf , determined by nephelometry , siemens healthcare diagnostics , munich , germany , normal range < 20  iu / ml ) , anti - ccp ab ( measured by elisa , aesku diagnostics , wendelsheim , germany , normal range < 18  u / ml ) , and antinuclear antibody ( ana , measured by elisa , aesku diagnostics , wendelsheim , germany , normal range < 1.00  s / co value ) . sigg and sigg4 levels were determined by immunonephelometry with bn prospec system ( dade behring , deerfield , il , usa ) using the following kits : n as igg and n latex igg4 ( siemens healthcare diagnostics products gmbh , marburg , germany ) . closed parker - pearson needle synovial biopsy was performed on knees of 46 ra patients among the above patients at enrollment . at least 6 pieces of synovial tissue were obtained per patient to minimize sampling error . sections ( 5  m ) were cut serially and mounted on adhesive glass slides . serial sections of synovial tissues were stained with hematoxylin and eosin and a 3-step immunoperoxidase method which was shown in detail in our previous study . serial sections were stained with rabbit anti - human igg antibody ( cell marque corporation , california , usa ) , rabbit anti - igg4 monoclonal antibody ( clone ep138 , epitomics , inc . , burlingame , usa ) , and the following commercial antibody preparations ( invitrogen , carlsbad , ca , usa ) : anti - cd38 ( clone spc32 , plasma cells ) , anti - cd3 ( clone ps1 , t cells ) , anti - cd20 ( clone l26 , b cell ) , anti - cd68 ( clone kp1 , macrophage - like synoviocytes and macrophages ) , and anti - cd34 ( clone qbend/10 , vascular endothelial cells ) . tonsil tissues were used as positive control tissues in each staining run . only tissue pieces containing integrated synovial histological synovitis was graded by two independent observers ( le - feng chen and ying - qian mo ) according to a three - component synovitis score   including enlargement of synovial lining layer , density of sublining resident cells , and inflammatory infiltration . each feature was scored from 0 to 3 and the sum provided the synovitis score from 0 to 9 . the densities of sublining igg- or igg4-positive plasma cells were evaluated under microscope and photographed with the matching microscope imaging software ( leica dm2500 , las v3.6 , leica microsystems gmbh , wetzlar , germany ) . since each photo revealed a synovial area of 0.11740  mm , the densities of igg4- or igg - positive plasma cells were counted manually in 9 photos with total area of nearly 1  mm by two independent observers ( le - feng chen and jian - da ma ) and the results were showed as cells per mm . the densities of cd38 , cd3 , cd20 , cd68 positive - staining cells , or cd34-positive vessels were also counted as mentioned above and showed as cells or vessels per mm . data were described with mean  standard deviation ( range ) or number ( percentage ) unless stated otherwise . mann - whitney rank - sum test was used for comparison between two groups and kruskal - wallis one - way analysis of variance on ranks among three or more groups . spearman 's rank order correlation test was used to assess the correlation between two variables . none of the patients had an overlap of igg4-rd with ra . among 136 ra patients , the mean of sigg4 was 1.52  1.27  g / l ( range 0.04~5.92 patients were then divided into elevated sigg4 group ( n = 62 ) and normal sigg4 group ( n = 74 ) . sigg4 level correlated positively with sigg level ( r = 0.424 , p < 0.001 ) . the mean sigg4/sigg ratio of 136 ra patients was 10%  7% ( range 0.3%~37% ) . the mean sigg4/sigg ratio of elevated sigg4 group was 16%  7% , which was significantly higher than that of normal sigg4 group ( 5%  3% ; p < 0.001 ) . ninety - five percent ( 59/62 ) in elevated sigg4 group and only 14% ( 10/74 ) in normal sigg4 group had elevated sigg4/sigg ratio (  = 89.976 , p < 0.001 ) . thirty percent ( 41/136 ) of all ra patients had never taken any disease modifying antirheumatic drugs ( dmards ) or corticosteroid before enrollment , and demographic characteristics of these patients are showed in table 1 . g / l , which was significantly higher than that of the treated patients ( 1.39  1.20 g / l ; p = 0.044 , figure 1 ) . the mean sigg4/sigg ratio of the untreated patients was 11%  7% ( range 0.4%~30% ) and 63% had elevated sigg4/sigg ratio . among 136 ra patients , 16% were in disease remission ( das28 < 2.6 ) , 10% in low disease activity ( lda , das28  2.6 to < 3.2 ) , 32% in moderate disease activity ( mda , das28  3.2 to 5.1 ) , and 41% in high disease activity ( hda , das28 > 5.1 ) . the mean sigg4 levels of patients in remission , lda , mda , or hda groups were 0.94  0.77 g / l , respectively , which showed significant difference among these four groups (  = 8.456 , p = 0.037 ) . mann - whitney rank - sum test was used for comparison between two groups for 6 times since there were 6 pairs among four groups and p < 0.0083 ( 0.05/6 ) was considered statistically significant . multiple comparisons revealed that sigg4 of hda group was significantly higher than that of remission group ( p = 0.003 ) . the ratios of patients with elevated sigg4 in remission , lda , mda , or hda groups were 32% ( 7/22 ) , 43% ( 6/14 ) , 43% ( 19/44 ) , and 54% ( 30/56 ) , respectively , showing no significant difference among these four groups ( p > 0.05 ) . patient global assessment of disease activity ( ptga ) , provider global assessment of disease activity ( prga ) , crp , esr , and ige of the elevated sigg4 group were significantly higher than those of the normal sigg4 group ( ptga , 6  3 versus 4  3 ; prga , 5  3 versus 4  3 ; crp , 38  42 mg / l versus 25  33  mg / l ; esr , 70  42  mm / h versus 48  32  mm / h ; ige , 604  1938  iu / ml versus 159  275  iu / ml ; all p < 0.05 ) . sigg4 level correlated positively but slightly with 28 tender joint counts ( 28tjc , r = 0.191 ) , health assessment questionnaire score ( haq , r = 0.221 ) , ptga ( r = 0.241 ) , prga ( r = 0.248 ) , crp ( r = 0.373 ) , esr ( r = 0.389 ) , ige ( r = 0.328 ) , and das28 ( r = 0.253 ; all p < 0.05 ) . among 41 untreated patients , no one was in disease remission , 7% were in lda , 37% were in mda , and 56% were in hda . the mean sigg4 levels of patients in lda , mda , or hda groups were 1.41  0.82 g / l , respectively , showing no significant difference among these three groups ( p > 0.05 ) . crp level of the elevated sigg4 group ( n = 23 ) was significantly higher than that of the normal sigg4 group ( n = 18 ; 44  31  mg / l versus 29  33  mg / l , p = 0.022 ) . sigg4 level correlated positively with crp ( r = 0.426 ) and esr ( r = 0.315 ; both p < 0.05 ) . rf and anti - ccp ab levels of the elevated sigg4 group were significantly higher than those of the normal sigg4 group ( rf , 513  636 iu / ml versus 245  392  iu / ml ; anti - ccp ab , 256  243  u / ml versus 162  199  u / ml ; both p < 0.05 ) , but there was no significant difference of ana between these two groups ( 1.2  1.2  s / co versus 1.1  0.9  s / co , p > 0.05 ) . sigg4 level correlated positively with rf ( r = 0.355 , p < 0.001 ) and anti - ccp ab ( r = 0.224 , p = 0.009 ) , but there was no significant correlation between sigg4 and ana ( p > 0.05 ) . the positive rate of rf in elevated sigg4 group ( 89% ) was significantly higher than that in normal sigg4 group ( 73% ;  = 5.250 , p = 0.022 ) , but there were no significant differences of positive rate of anti - ccp ab or ana between these two groups ( both p > 0.05 ) . seventy - five ra patients with active disease at baseline completed 6-month follow - up . rf and anti - ccp ab levels of 67 patients were determined again at 6-month visit , and the results showed that rf and anti - ccp ab levels significantly decreased after treatment both in elevated baseline sigg4 group ( n = 28 ) and in normal baseline sigg4 group ( n = 39 , all p < 0.05 ) . reduction range of rf level after treatment in elevated baseline sigg4 group was significantly larger than that in normal baseline sigg4 group ( p < 0.01 , table 2 ) . among the 75 patients who completed 6-month follow - up , 45 patients received methotrexate ( mtx ) + leflunomide ( lef ) therapy , 18 received mtx + tumor necrosis factor ( tnf)- antagonist ( 12 weeks ) , and 12 received other dmard(s ) therapies . for patients receiving mtx + lef , 50% ( 9/18 ) of patients with elevated baseline sigg4 were good responders who reached therapeutic target of das28 < 3.2 at 6-month visit , which was significantly lower than 85% ( 23/27 ) of patients with normal baseline sigg4 (  = 6.508 , p = 0.011 ) . for patients receiving mtx + tnf- antagonist , there was no significant difference in the percentage of good responders between patients with elevated baseline sigg4 and normal baseline sigg4 , 70% ( 7/10 ) versus 88% ( 7/8 ) , p > 0.05 . among 46 ra patients receiving synovial biopsy , 78% of them were female and the mean age was 55  14 years , mean disease duration was 74  75 months , and the mean das28 was 5.0 ( range 1.2~8.2 ) . as shown in figure 2 , cytoplasmic expression of igg4 was extremely distributed in synovial sublining area . the igg4-positive cells showed typical morphology of plasma cells and ihc staining of cd3 , cd20 , cd38 , or cd68 on serial slides confirmed that igg4 is mainly expressed in the cd38-positive plasma cells . the mean count of synovial igg4-positive plasma cells was 155  175/mm ( range 0~735 ) . igg - positive cells were defined as clear - cut cells with strong cytoplasmic reactivity of igg . the mean count of synovial igg - positive plasma cells was 555  474/mm ( range 25~2020 ) . spearman 's rank order correlation test showed synovial igg4-positive plasma cells correlated positively and significantly with total synovitis score ( r = 0.374 , p = 0.010 ) , inflammatory infiltration subscore ( r = 0.335 , p = 0.023 ) , and density of resident cells subscore ( r = 0.364 , p = 0.013 ) . patients were divided into severe inflammatory group ( inflammatory infiltration subscore : 2~3 , n = 31 ) and mild inflammatory group ( 0~1 , n = 15 ) . patients in severe inflammatory group had higher igg4-positive plasma cells than those in mild inflammatory group ( 202  189/mm versus 49  55/mm , figure 3(b ) ) . positive and significant correlation was found between igg4-positive plasma cells and cd3-positive ( figure 3(c ) ) , cd20-positive ( figure 3(d ) ) , or cd38-positive cell counts ( figure 3(e ) ) , but not cd68-positive cell count or cd34-positive vessel count ( both p > 0.05 ) . sigg4 correlated positively and significantly with synovial igg4-positive plasma cells ( figure 3(a ) ) , but no significant correlation of sigg4 with total synovitis score or subscores was found . this study detected sigg4 level of 136 ra patients and 46% of them had elevated sigg4 , which was disproportional to total sigg . we first demonstrated that ra with elevated sigg4 may be a specific clinical phenotype characterized by higher disease activity , higher level of autoantibodies , and poor response to mtx + lef therapy . further immune pathological study in synovium of 46 ra patients first demonstrated that elevated sigg4 correlated with increased synovial igg4-positive plasma cells , which correlated with histological synovitis in ra . elevated sigg4 is not specific to igg4-rd , although it is an important characteristic of igg4-rd . patients with allergic disorders , parasite infestations , pemphigus , castleman 's disease , or churg - strauss syndrome   may have elevated sigg4 . lin and li   found that four subclasses of igg ( igg1~igg4 ) in serum were , respectively , significantly higher in 72 ra patients than that in healthy people , although sigg4 remained the lowest subclasses . in this study , we found that 46% of ra patients had elevated sigg4 determined by immunonephelometry . synthesis of igg4 in vitro was usually regulated by certain cytokines and chemokines such as il-10 and il-6 [ 1719 ] . il-10 , a lymphokine with important anti - inflammatory property , enhanced il-4-induced igg4 switching . serum il-10 was elevated in igg4-related pancreatitis , cholangitis , or tubulointerstitial nephritis , supporting that il-10 might participate in the sigg4 elevation and igg4-positive plasma cell infiltration in igg4-rd . serum il-10 was also reported to be higher in ra patients compared to healthy controls , but il-6 inducing igg4 elevation might be partly through il-21 expressed in cd4 + t cells , which promotes differentiation of b cells into antibody - secreting plasma cells . elevated il-6 level has been found in serum or synovium of ra patients [ 25 , 26 ] , but not in igg4-rd [ 27 , 28 ] . il-6 is a key proinflammatory cytokine in ra and plays important roles in the regulation of the immune response , inflammation , hematopoiesis , and bone metabolism . sigg4 decreased in 7 of 8 ra patients who received treatment of tocilizumab , a monoclonal antibody to the il-6 receptor , indicating that il-6 may be the principal cytokine that induces igg4 elevation in ra . eight percent were reported as cutoff point of sigg4/sigg ratio to discriminate igg4-rd from other mimickers with specificity varying from 59%   to 87.5% . in this study , we found that sigg4/sigg ratio of ra patients with normal sigg4 group was 5%  3% , which was similar with 3%~7% reported in healthy people . our study also showed that sigg4/sigg ratio of ra patients with elevated sigg4 group was 16%  7% , which was significantly higher than that of normal sigg4 group , suggesting sigg4 increased disproportionally to total sigg in ra patients with elevated sigg4 . autoantibodies such as rf , anti - ccp ab , have igg type which included igg1~igg4 subclasses . .   showed that rf - igg1 was the most prevalent subclass of rf - igg in ra patients , followed by rf - igg4 which had higher level than rf - igg2 or -igg3 . similarly , anti - ccp ab - igg4 was also the second subclass of anti - ccp ab - igg in ra patients . our study demonstrated that ra patients with elevated sigg4 had significantly higher rf and anti - ccp ab levels at baseline , as well as higher reduction of rf after treatment , indicating that ra patients with elevated sigg4 may have more igg4 autoantibodies such as rf - igg4 which was easy to decline during treatment . despite relative lack of study on the change of rf - igg subclasses including rf - igg4 , there were studies on the change of different anti - ccp ab - igg subclasses in ra patients during biological dmards therapy . reported that anti - ccp ab - igg4 , but not anti - ccp ab - igg1 , reduced significantly after 6-month tocilizumab treatment in ra patients . bos et al .   reported that both anticitrullinated protein antibody- ( acpa- ) igg1 and igg4 of ra patients reduced significantly after 46-week infliximab or 28-week adalimumab treatment , accompanied by significant reduction of acpa - igg4/igg1 ratio , indicating preferential decrease in acpa - igg4 . our study did not find significant reduction of anti - ccp ab between ra patients with elevated sigg4 and normal sigg4 , perhaps due to different therapeutic regimes or predominant use of traditional dmards or the small number of patients . for autoimmune pancreatitis , patients with elevated sigg4 may be prone to systemic disease with high disease severity , and both sigg4 and sigg4/sigg ratio significantly decreased after glucocorticoid therapy [ 3638 ] . yamamoto et al .   demonstrated that 17% ( 5/29 ) of ra patients with active disease ( das28 : 5.1  1.2 ) at onset had elevated sigg4 level . in this study , we included 22 ra patients with disease remission and divided patients into different extents of disease activity to evaluate the correlation of sigg4 with ra disease activity . our result showed that sigg4 of ra patients with hda was significantly higher than those with disease remission . further comparative analysis and correlation test confirmed that ra disease activity indexes including esr , crp , and das28 positively correlated with sigg4 . the untreated patients were analyzed separately and only positive correlation between sigg4 and crp or esr was found . no correlation of sigg4 with das28 in the untreated patients may be explained by bias of patient enrollment , since 93% of the untreated patients were in mda or hda . our result also showed that only 50% of patients with elevated baseline sigg4 reached das28 < 3.2 after 6-month mtx + lef combination therapy , significantly lower than 85% of patients with normal baseline sigg4 . these results suggest that sigg4 has correlation with clinical synovitis which determines clinical disease activity of ra patients . marked igg4-positive plasma cells infiltration and dense lymphoplasmacytic infiltration are both important histological features of igg4-rd , which also appear in ra synovium . this study demonstrated that marked igg4-positive plasma cells infiltrated in rheumatoid synovium and correlated with total synovitis score , inflammatory infiltration subscore , cd3-positive t cells , cd20-positive b cells , or cd38-positive plasma cells . sigg4 also correlated positively with igg4-positive plasma cells , but not with total synovitis score or its subscores , maybe due to the small number of patients . fibrosis is another vital histological feature of igg4-rd , especially storiform fibrosis which resembles the spokes of a cartwheel with spindle cells radiating from a center . our result showed fibrosis was also present in ra synovium . transforming growing factor ( tgf)- , a powerful fibrogenic cytokine , was upregulated and might induce fibrosis in the involved organ(s ) of igg4-rd . fibrosis of ra synovium may also be induced by tgf- which was also upregulated in ra synovium . however , further studies are needed to elucidate the pathogenetic role of synovial igg4 and its correlation with fibrosis in ra . igg4 may be an anti - inflammatory molecule for fab ( fragment antigen binding ) arm exchange . one igg4 swap a heavy chain and attached light chain ( half - molecule ) with a heavy - light chain pair from another igg molecule , which results in bispecific antibodies without ability of crosslink antigens or form immune complexes . additionally , fc ( fragment crystallizable ) of igg4 interacted with fc of other subclasses of igg to dampen the inflammatory response of these igg molecules . for instance , for patients with pemphigus foliaceus , an autoimmune blistering skin disease , igg4 is the predominant antibody against desmoglein 1 and mediates the formation of cutaneous blisters [ 43 , 44 ] . thus , further studies are needed to clarify the exact pathogenetic role of igg4 in ra . our results showed that elevated sigg4 in ra is common and disproportional to total igg and ra with elevated sigg4 may be a specific clinical phenotype with higher disease activity , higher level of autoantibodies , and poor response to mtx + lef therapy .
objectives . to explore the correlation of serum igg4 ( sigg4 ) with clinical manifestations or therapeutic response in rheumatoid arthritis ( ra ) . methods . 
 consecutive 136 ra patients were recruited and followed up at regular interval . 
 sigg4 was detected by immunonephelometry . 
 serial synovial tissue sections from 46 ra patients were stained immunohistochemically for igg4 . 
 results . 
 forty - six percent of 136 ra patients had elevated sigg4 . 
 patients with elevated sigg4 had higher sigg4/sigg ratio , c - reactive protein , erythrocyte sedimentation rate , rheumatoid factor , and anticyclic citrullinated peptide antibodies than those with normal sigg4 ( all p < 0.05 ) . among 45 patients who received methotrexate and leflunomide therapy , 50% ( 9/18 ) of patients with elevated sigg4 and 85% ( 23/27 ) of patients with normal sigg4 reached therapeutic target ( disease activity score of 28 joints < 3.2 ) at 6-month visit ( 2 = 6.508 , p = 0.011 ) . 
 igg4-positive plasma cell count correlated positively with sigg4 , total synovitis score , and cd3- , cd20- , and cd38-positive cell counts ( all p < 0.05 ) . 
 conclusions . 
 our results showed that elevated sigg4 in ra is common and disproportional to total igg and ra with elevated sigg4 may be a specific clinical phenotype with higher disease activity , higher level of autoantibodies , and poor response to methotrexate and leflunomide therapy .