ra is also characterized by autoantibody production ( antibody of anti - citrullinated protein and rheumatoid factor ) , hyperplasia and synovial inflammation , and systemic features ( e.g. , pulmonary , cardiovascular , psychological , and skeletal disorders ) .
even though some patients have mild self - limited disease , many of them have experienced joint destruction , severe physical disability , and multiple co - morbidities [ 57 ] .
unfortunately , patients with ra may develop various systemic symptoms such as fatigue , fever , anemia , weight loss , anorexia , muscle weakness , and osteoporosis .
death rates are more than twice as high in ra patients as in the general population , and this gap seems to be widening .
the specific cause of ra is unknown , but it has been suspected that a complex interplay among environmental factors , genotype , and chance contributes to the occurrence of ra .
unrestrained cytokine production has been correlated with various diseases , including ra . in ra ,
chronic inflammation in ra is induced by the imbalance between cytokines of pro- and anti - inflammation and the induction of autoimmunity .
potential triggers of ra include autoantibodies such as anti - citrullinated peptides antibodies and rheumatoid triggers , and pro - inflammatory cytokines like tumor necrosis factor- ( tnf- ) and interleukin 6 ( il-6 ) .
cytokines modulate a large range of inflammatory processes correlated with the pathogenesis of ra , and appear abundantly in the serum and the arthritic synovial fluid of patients with ra .
after synovitis or synovial inflammation develops from autoimmunity in ra , additional triggers , including the production of pro - inflammatory cytokines ( e.g. , tnf- and il-6 ) , further develop bone erosion and osteoclastogenesis , which relates to the regression of bone - resorbing osteoclasts .
tnf- , secreted by activated macrophages and t - cells , exerts pro - inflammatory effects through binding to one of its receptors , p55 ( tnf - ri ) or p75 ( tnf - rii ) , and plays a vital role in production of other cytokines and the induction of chronic inflammation .
il-6 , as another pro - inflammatory cytokine , drives the activation of local synovial leukocyte and antibody production .
evidence from previous studies suggest that tnf- and il-6 play a significant role in the occurrence and development of ra due to their pro - inflammatory effects .
however , studies have also indicated that il-6 and tnf- can have anti - inflammatory functions under some circumstances .
consequently , the purpose of our meta - analysis was to investigate the association between ra and serum levels of il-6 and tnf- via a meta - analysis .
electronic databases  ebsco , pubmed , ovid , springerlink , web of science , wiley , wanfang data , and china national knowledge infrastructure ( cnki)were searched to collect all relevant published studies ( last updated search in october , 2014 ) . the following key words and subject terms were used in the searches : ra , il-6 , and tnf-. we also selected (  tumor necrosis factor - alpha  or 
tnf - alpha  or  tumor necrosis factor  or  tnf  ) , (  interleukin-6  or 
il-6  or  differentiation factor-2 , b - cell  or  b cell stimulatory factor-2  ) , (  arthritis , rheumatoid  or  rheumatoid arthritis  or  ra  ) in the search strategy .
studies were qualified for inclusion if they met the following criteria : ( 1 ) the study type was case - control ; ( 2 ) the research topic mentioned the association between ra and serum levels of il-6 and tnf- ; ( 3 ) all subjects were patients clinically diagnosed with ra ; ( 4 ) the detection method was enzyme - linked immunosorbent assay ( elisa ) ; and ( 5 ) complete data was supplied in included studies . when the extracted studies were published by the same authors , only the complete or latest study was included . studies were excluded if : ( 1 ) data were incomplete ; ( 2 ) the differences in baseline characteristics between the case group and the control group were too great ; ( 3 ) the article was published repeatedly ; or ( 4 ) the diagnostic criteria for study subjects were ambiguous .
relevant data eligible for the final analyses in retrieved papers were extracted by 2 investigators separately on the basis of the inclusion criteria , including surname of first author , country of origin , publication year , ethnicity , language , disease , detection method , age , sex , study design , and sample size .
review reports from the 2 investigators were compared to identify inconsistency , and disputes were settled through further discussion and reexaminations among all investigators .
the methodological quality of the included cases was evaluated by more than 2 investigators under the methodological index for non - randomized studies ( minors ) criteria .
minors is an effective scoring tool , with a 12-item assessment , and each item can be scored from 0 to 2 , with an ideal score of 16 for non - comparative studies and a score of 24 for comparative studies .
the details of 12 criteria were displayed : the aim was clearly stated ( minors 01 ) , consecutive inclusion of patients or not ( minors 02 ) , collection of prospective data or not ( minors 03 ) , the endpoints were appropriate for the aim of the study ( minors 04 ) , unbiased assessment of endpoint or not ( minors 05 ) , appropriate follow - up period or not ( minors 06 ) , less than 5% loss of follow - up or not ( minors 07 ) , prospectively calculated study size or not ( minors 08 ) , adequate control group or not ( minors 09 ) , contemporary groups or not ( minors 10 ) , equivalent baseline of groups or not ( minors 11 ) , and adequate statistical analyses or not ( minors 12 ) .
stata 12.0 software ( stata corp , college station , tx , usa ) was used for statistical analyses .
the differences of serum levels of inflammatory cytokines ( il-6 and tnf- ) between the case and control groups were estimated by the standardized mean difference ( smds ) with 95% confidence interval ( ci ) .
cochran s q statistic ( p<0.05 was considered significant ) and the i test ( 0% , no heterogeneity ; 100% , maximal heterogeneity ) were also used to reflect the heterogeneity among studies .
there was great heterogeneity among studies at p<0.05 or i > 50% ; therefore , a random - effects model was used ; otherwise , a fixed - effects model was used .
univariate and multiple meta - regression analyses were conducted to evaluate the potential source of heterogeneity , and monte carlo simulation ( mcs ) was used to correct and verify the result .
sensitivity analysis was achieved by deleting each included study one at a time to assess the effect of each study on the overall outcome .
a funnel plot was constructed to evaluate publication bias that might influence the reliability of the results .
electronic databases  ebsco , pubmed , ovid , springerlink , web of science , wiley , wanfang data , and china national knowledge infrastructure ( cnki)were searched to collect all relevant published studies ( last updated search in october , 2014 ) . the following key words and subject terms were used in the searches : ra , il-6 , and tnf-. we also selected (  tumor necrosis factor - alpha  or 
tnf - alpha  or  tumor necrosis factor  or  tnf  ) , (  interleukin-6  or 
il-6  or  differentiation factor-2 , b - cell  or  b cell stimulatory factor-2  ) , (  arthritis , rheumatoid  or  rheumatoid arthritis  or  ra  ) in the search strategy .
studies were qualified for inclusion if they met the following criteria : ( 1 ) the study type was case - control ; ( 2 ) the research topic mentioned the association between ra and serum levels of il-6 and tnf- ; ( 3 ) all subjects were patients clinically diagnosed with ra ; ( 4 ) the detection method was enzyme - linked immunosorbent assay ( elisa ) ; and ( 5 ) complete data was supplied in included studies . when the extracted studies were published by the same authors , only the complete or latest study was included .
studies were excluded if : ( 1 ) data were incomplete ; ( 2 ) the differences in baseline characteristics between the case group and the control group were too great ; ( 3 ) the article was published repeatedly ; or ( 4 ) the diagnostic criteria for study subjects were ambiguous .
relevant data eligible for the final analyses in retrieved papers were extracted by 2 investigators separately on the basis of the inclusion criteria , including surname of first author , country of origin , publication year , ethnicity , language , disease , detection method , age , sex , study design , and sample size .
review reports from the 2 investigators were compared to identify inconsistency , and disputes were settled through further discussion and reexaminations among all investigators .
the methodological quality of the included cases was evaluated by more than 2 investigators under the methodological index for non - randomized studies ( minors ) criteria .
minors is an effective scoring tool , with a 12-item assessment , and each item can be scored from 0 to 2 , with an ideal score of 16 for non - comparative studies and a score of 24 for comparative studies .
the details of 12 criteria were displayed : the aim was clearly stated ( minors 01 ) , consecutive inclusion of patients or not ( minors 02 ) , collection of prospective data or not ( minors 03 ) , the endpoints were appropriate for the aim of the study ( minors 04 ) , unbiased assessment of endpoint or not ( minors 05 ) , appropriate follow - up period or not ( minors 06 ) , less than 5% loss of follow - up or not ( minors 07 ) , prospectively calculated study size or not ( minors 08 ) , adequate control group or not ( minors 09 ) , contemporary groups or not ( minors 10 ) , equivalent baseline of groups or not ( minors 11 ) , and adequate statistical analyses or not ( minors 12 ) .
stata 12.0 software ( stata corp , college station , tx , usa ) was used for statistical analyses .
the differences of serum levels of inflammatory cytokines ( il-6 and tnf- ) between the case and control groups were estimated by the standardized mean difference ( smds ) with 95% confidence interval ( ci ) .
cochran s q statistic ( p<0.05 was considered significant ) and the i test ( 0% , no heterogeneity ; 100% , maximal heterogeneity ) were also used to reflect the heterogeneity among studies .
there was great heterogeneity among studies at p<0.05 or i > 50% ; therefore , a random - effects model was used ; otherwise , a fixed - effects model was used .
univariate and multiple meta - regression analyses were conducted to evaluate the potential source of heterogeneity , and monte carlo simulation ( mcs ) was used to correct and verify the result .
sensitivity analysis was achieved by deleting each included study one at a time to assess the effect of each study on the overall outcome .
a funnel plot was constructed to evaluate publication bias that might influence the reliability of the results .
a total of 849 studies were chosen from the 8 databases via screening both title and key words . followed by removing reviews , letters , meta - analyses ( n=5 ) , duplicates ( n=21 ) , non - human studies ( n=14 ) , and studies not relevant to the research topics ( n=760 ) , the remaining studies ( n=49 ) were checked and an additional 31 studies were excluded because they were not case - control or cohort studies ( n=7 ) , not related to tnf- ( n=12 ) , or not relevant to il-6 ( n=12 ) . after the remaining 18 studies were further reviewed , 14 studies [ 23,3244 ] were enrolled in the analysis . during the final selection process ,
there were 890 patients with ra in the case group and 441 controls in this meta - analysis .
heterogeneity was found among included studies according to the heterogeneity test ( i=97.6% , p<0.001 ) ; thus , a random - effects model was used .
the results of this meta - analysis indicated that the serum il-6 level of ra patients was evidently higher than in the controls , suggesting a statistically significant difference between the case and control groups ( smd=2.40 , 95% ci=1.57~3.24 , p<0.001 ) ( figure 1a ) . in ethnic subgroups ,
the serum il-6 level of patients with ra was also significantly higher compared with the controls in asians and caucasians ( asians : smd=3.64 , 95% ci=2.16~5.12 , p<0.001 ; caucasians : smd=0.75 , 95% ci=0.47~1.02 , p<0.001 ) ( figure 2a ) .
the result of univariate meta - regression analysis suggests that ethnicity might be the source of heterogeneity ( p=0.036 ) , but the year of publication , language , and sample size were not associated with the heterogeneity ( year of publication : p=0.504 ; language : p=0.318 ; sample size : p=0.545 ) as presented in figure 3a3d .
nevertheless , in multiple meta - regression analysis , ethnicity , year of publication , sample size , and language were not potential sources of heterogeneity ( table 1 ) .
a random - effects model was used due to heterogeneity among included studies ( i=96.8% , p<0.001 ) according to the heterogeneity test . as presented in figure 1b
, the results indicated that the serum tnf- level of patients with ra was clearly higher than in the controls , and there was a statistically significant difference between the case and control groups ( smd=1.93 , 95% ci=1.23~2.64 , p<0.001 ) . on the basis of analyses of the ethnic subgroups , the serum tnf- level of ra patients was also significantly higher compared with the controls in asians and caucasians ( asians : smd=2.74 , 95% ci=1.58~3.91 , p<0.001 ; caucasians : smd=0.81 , 95% ci=0.50~1.11 , p<0.001 ) ( figure 2b ) .
the univariate meta - regression analysis suggested that the main source of heterogeneity might be ethnicity ( p=0.016 ) , but not the year of publication , language , or sample size ( year of publication : p=0.660 ; language : p=0.079 ; sample size : p=0.899 ) as shown in figure 3e3h .
additionally , in multiple meta - regression analysis , ethnicity , year of publication , language , and sample size were not sources of heterogeneity ( table 2 ) .
the sensitivity analysis suggests that all included studies had no evident influence on the pooled smds of serum il-6 and tnf- level in ra patients and the controls , and the shapes of funnel plots of the differences in serum level of il-6 and tnf- showed asymmetry ( figure 4 ) .
thus , egger s test was used to further provide statistical evidence of funnel plot asymmetry , and also showed publication bias among included studies ( il-6 : p<0.001 ; tnf- : p=0.007 ) .
a total of 849 studies were chosen from the 8 databases via screening both title and key words . followed by removing reviews , letters , meta - analyses ( n=5 ) , duplicates ( n=21 ) , non - human studies ( n=14 ) , and studies not relevant to the research topics ( n=760 ) , the remaining studies ( n=49 ) were checked and an additional 31 studies were excluded because they were not case - control or cohort studies ( n=7 ) , not related to tnf- ( n=12 ) , or not relevant to il-6 ( n=12 ) . after the remaining 18 studies were further reviewed , 14 studies [ 23,3244 ] were enrolled in the analysis . during the final selection process ,
there were 890 patients with ra in the case group and 441 controls in this meta - analysis .
heterogeneity was found among included studies according to the heterogeneity test ( i=97.6% , p<0.001 ) ; thus , a random - effects model was used .
the results of this meta - analysis indicated that the serum il-6 level of ra patients was evidently higher than in the controls , suggesting a statistically significant difference between the case and control groups ( smd=2.40 , 95% ci=1.57~3.24 , p<0.001 ) ( figure 1a ) . in ethnic subgroups ,
the serum il-6 level of patients with ra was also significantly higher compared with the controls in asians and caucasians ( asians : smd=3.64 , 95% ci=2.16~5.12 , p<0.001 ; caucasians : smd=0.75 , 95% ci=0.47~1.02 , p<0.001 ) ( figure 2a ) .
the result of univariate meta - regression analysis suggests that ethnicity might be the source of heterogeneity ( p=0.036 ) , but the year of publication , language , and sample size were not associated with the heterogeneity ( year of publication : p=0.504 ; language : p=0.318 ; sample size : p=0.545 ) as presented in figure 3a3d .
nevertheless , in multiple meta - regression analysis , ethnicity , year of publication , sample size , and language were not potential sources of heterogeneity ( table 1 ) .
a random - effects model was used due to heterogeneity among included studies ( i=96.8% , p<0.001 ) according to the heterogeneity test . as presented in figure 1b
, the results indicated that the serum tnf- level of patients with ra was clearly higher than in the controls , and there was a statistically significant difference between the case and control groups ( smd=1.93 , 95% ci=1.23~2.64 , p<0.001 ) . on the basis of analyses of the ethnic subgroups , the serum tnf- level of ra patients was also significantly higher compared with the controls in asians and caucasians ( asians : smd=2.74 , 95% ci=1.58~3.91 , p<0.001 ; caucasians : smd=0.81 , 95% ci=0.50~1.11 , p<0.001 ) ( figure 2b ) .
the univariate meta - regression analysis suggested that the main source of heterogeneity might be ethnicity ( p=0.016 ) , but not the year of publication , language , or sample size ( year of publication : p=0.660 ; language : p=0.079 ; sample size : p=0.899 ) as shown in figure 3e3h .
additionally , in multiple meta - regression analysis , ethnicity , year of publication , language , and sample size were not sources of heterogeneity ( table 2 ) .
the sensitivity analysis suggests that all included studies had no evident influence on the pooled smds of serum il-6 and tnf- level in ra patients and the controls , and the shapes of funnel plots of the differences in serum level of il-6 and tnf- showed asymmetry ( figure 4 ) .
thus , egger s test was used to further provide statistical evidence of funnel plot asymmetry , and also showed publication bias among included studies ( il-6 : p<0.001 ; tnf- : p=0.007 ) .
one of the characteristics of ra was consistent chronic inflammation resulting in damage to synovial tissue , which led to irreversible tissue or joint damage .
it has been reported that the high level of inflammation in vivo is correlated with serious joint damage and the poor prognosis of ra . il-6 and tnf- are 2 well - known inflammatory cytokines with critical roles in modulating tissue inflammation ; moreover , the plasma concentrations of il-6 and tnf- can reflect the severity of inflammation in vivo .
we investigated the association between ra and the serum levels of il-6 and tnf- on the basis of previous studies .
we found that compared with the controls , the serum levels of il-6 and tnf- were significantly higher in patients with ra , suggesting that il-6 and tnf- might play important roles in the pathogenesis of ra . in ra , tnf- is secreted by various cell types , predominantly by macrophages and dendritic cells in reaction to the interactions between pathogen - associated molecular patterns ( pamps ) or damage - associated molecular patterns ( damps ) and pattern - recognition receptors ( prrs ) or to the cytokine environment .
il-6 is a multifunctional cytokine with biological activities that include the modulation of inflammation , immune response , and hematopoiesis .
varieties of innate effector cells , such as mast cells , macrophages , and natural killer cells , are discovered in the synovial membrane , but neutrophils reside mostly in synovial fluid .
granulocyte colony - stimulating factor , macrophage colony - stimulating factor , and granulocyte  macrophage colony - stimulating factor ( gm - csf ) promote the maturation of the above innate effector cells , trafficking to the synovium , and their efflux from the bone marrow .
importantly , macrophages act as central effectors of synovitis and are effective biologic agents that could reduce macrophage infiltration consistently in the synovium .
macrophages act via release of cytokines , for instance , tnf- and il-1 , -6 , -12 , -15 , -18 , and -23 , with tnf- and il-6 being the most predominant mediators , eventually resulting in the breakdown of extracellular matrix of bone and cartilage .
as the pathology of ra was unclear , most studies speculated that synovial hyperplasia and progressive joint destruction was involved in the possible mechanism of the immune system attacking the joints .
the inflammatory reaction involved in the synovial hyperplasia and joint destruction could be enhanced by il-6 , which amplifies the inflammatory cell infiltration . moreover , the synovial fibroblastic cells secreted il-6 once it was stimulated by inflammatory cytokines such as il-1 and tnf- and , in turn , il-6 enhanced the proliferation of synovial fibroblastic cells in the presence of soluble il-6 receptor .
the explanation could be that il-6 and tnf- affect the progression in synovial hyperplasia , resulting in development and progression of ra .
therefore , it was reasonable to think that the serum levels of il-6 and tnf- in ra patients were evidently higher compared with the controls , which is consistent with some previous studies that revealed that il-6 and tnf- may contribute to the development of ra due to their pro - inflammatory effects .
although ra was characterized by persistent synovitis and continuous joint destruction , anemia was the most common symptom during the early stage of the disease .
research evidence shows that il-6 injection contribute to induce of anemia , and vice versa , suggesting that il-6 , but not tnf- , plays a crucial role in anemia in monkey collagen - induced arthritis , whose pathogenesis was similar with ra , by inhibiting the il-6-induced hepcidin production . a study by godfrin - valnet et al .
identified a positive trend of il-6 and the serum c - terminal cross - linking telopeptide of type i collagen ( ctx ) levels in patients with osteoporosis , revealing that increased level of il-6 was correlated with accelerated bone resorption from increased osteoclastogenesis and reduced bone formation .
moreover , elevated vascular endothelial growth factor ( vegf ) level was found in ra patients , suggesting that vegf is involved in ra pathogenesis .
release of tnf- and il-6 from synovial tissue modifies the function of distant tissue , including vascular endothelial tissue , which could lead to insulin resistance , abnormal blood fats , and endothelial dysfunction . to avoid the influence of other factors on the overall results , we selected ethnic subgroups analyses to further validate our results .
we found that the serum il-6 and tnf- levels of ra patients were also significantly higher than in the controls in asians and caucasians , indicating that ethnicity did not affect our final results .
additionally , ethnicity , year of publication , language , and sample size also did not affect the results of this meta - analysis .
for instance , because the inclusion process was performed manually , there might be some deviation that could impact the results ; data on gender and age were lost in several included studies , which might be correlated with the serum levels of il-6 and tnf- and thereby might influence the result of this meta - analysis .
in addition , according to the baseline characteristics of included studies , the sample size was too small to make firm conclusions about the correlation between ra and serum levels of il-6 and tnf-.
il-6 and tnf- , as 2 inflammatory cytokines , might play a vital role in the pathogenesis of ra through their pro - inflammatory effects .
 backgroundwe aimed to investigate the association of rheumatoid arthritis ( ra ) with interleukin 6 ( il-6 ) and tumor necrosis factor- ( tnf- ) through a meta-analysis.material/methodsthe case - control studies that investigated the association between ra and serum levels of il-6 and tnf- were retrieved strictly according to the inclusion and exclusion criteria . 
 the statistical analysis was performed using stata statistical software ( version 12.0 , stata corporation , college station , tx , usa).resultsfourteen studies were enrolled in our meta - analysis , with a total of 890 patients with ra and 441 healthy people as the controls . 
 the results of this meta - analysis revealed that the serum il-6 and tnf- levels of ra patients were significantly higher than in the controls , and this difference was statistically significant ( il-6 : smd=2.40 , 95% ci=1.57~3.24 , p<0.001 ; tnf- : smd=1.93 , 95% ci=1.23~2.64 , p<0.001 ) . according to ethnic subgroup analysis , the serum il-6 and tnf- levels of ra patients were also significantly higher compared with the controls in asians and caucasians ( il-6 : asians : smd=3.64 , 95% ci=2.16~5.12 , p<0.001 ; caucasians : smd=0.75 , 95% ci=0.47~1.02 , p<0.001 ; tnf- : asians : smd=2.74 , 95%ci=1.58~3.91 , p<0.001 ; caucasians : smd=0.81 , 95% ci=0.50~1.11 , p<0.001).conclusionsil-6 and tnf- may play crucial roles in the activity and severity of ra . 
Background
Material and Methods
Search strategy
Inclusion and exclusion criteria
Data extraction and quality evaluation
Statistical analysis
Results
The baseline characteristics of included studies
Differences in serum levels of IL-6
Differences in serum levels of TNF-
Sensitiveness analysis and publication bias
Discussion
Conclusions