rheumatoid arthritis ( ra ) is a chronic , progressive , inflammatory autoimmune disease in which the body 's immune system mistakenly attacks the joint .
the disease produces an inflammatory infiltrate of immune cells as well as a series of destructive events such as synovial hyperplasia , pannus setting , bone and cartilage erosion , and joint destruction .
it results in swelling and pain in the joints and around them [ 1 , 2 ] . in ra ,
activation of innate immunity in early disease , followed by the appearance of adaptive immune responses ultimately leads to a destructive phase .
the pathophysiology of ra implies the existence of t and b cells , various immune modulators ( cytokines and effector cells ) , and signalling pathways .
the complex interaction of immune modulators causes joint damage starting at the synovial membrane and covering most structures .
this disequilibrium between pro- and anti - inflammatory cytokine activities facilitates the induction of autoimmunity , chronic inflammation , and joint damage .
it is less known , though , how cytokines are organized within a hierarchical regulatory network and which cytokines may qualify as best targets for clinical intervention a priori [ 35 ] .
ra pathogenesis is caused by b cells not only through antigen presentation , but also through the production of antibodies , autoantibodies , and cytokines [ 2 , 5 ] .
there are no disease - specific diagnostic features in ra and patients can have a wide range of manifestations .
the diagnosis of ra is given by a combination of symptoms , signs , serologic tests , and radiologic findings , as established by the american college of rheumatology .
since early inflammatory arthritis is a clinically heterogeneous disease , cytokine networks are known to play a critical role in the pathogenesis of rheumatoid arthritis ; a panel of pro- and anti - inflammatory cytokines and associated autoantibodies were measured to identify the biologically based subsets of early rheumatoid arthritis ( era ) .
it requires assessment , not only of the current clinical picture but also of the potential for change .
therefore , identifying the early rheumatoid arthritis ( ra ) is a crucial step in controlling the progress of the disease .
. early diagnosis of rheumatoid arthritis ( ra ) is essential , because there is strong evidence that early treatment with one or more disease - modifying antirheumatic drugs improves the evolution [ 10 , 11 ] .
when dmards therapy is introduced early in patients , function and radiological outcome in the long term are better than cases when it is delayed .
our research objectives were to assess the concentrations of il-13 and il-17 in serum of patients with era , the investigation of correlation between the concentrations of these cytokines and disease activity score and the concentration of some autoantibodies in relation to the control group , and the evaluation of the utility of il-13 and il-17 concentration measurements as markers of disease activity .
we accomplished a study which included 30 patients diagnosed with early rheumatoid arthritis , gender ratio 6 m/24 f , and mean age 56.22 years ; in parallel , we investigated a control group that included 28 persons unaffected by early rheumatoid arthritis or other diseases . controls were matched for sex , age at the time point of blood sampling , and area of residence ( rural or urban ) .
early ra patients fulfilled the american college of rheumatology ( acr ) 1987 revised criteria for the classification of ra .
they were all investigated , diagnosed , and included in the studied group , following the revised classification criteria of the american college of rheumatology , in 2010 .
all patients accomplished the inclusion criteria for early rheumatoid arthritis ( two or more swollen joints dating more than 2 weeks , but less than 12 months from onset ) .
we excluded , from the start of the study , patients with other autoimmune diseases , those who received treatment with dmards , glucocorticoids , or / and vitamins , the women during pregnancy , and persons with diabetes mellitus or metabolic syndrome . the study cohort comprised patients firstly evaluated for early arthritis because we aimed to investigate era patients to find a better and faster way of discrimination between affected and unaffected cases . based on the das28 results ,
the 30 era patients were subdivided into three groups : mild ( 2.6 < das28  3.2 ) , moderate ( 3.2 < das28  5.1 ) , and severe ( 5.1 < das28 ) .
serum samples were collected from 30 patients and from 28 controls ( healthy persons ) and analyzed for concentrations of il-13 and il-17 , anticyclic citrullinated peptide antibodies ( anti - ccp ) , rheumatoid factor igm isotype ( igm - rf ) , anti - cardiolipin igg isotype ( igg - acl ) , anti - ra33 , erythrocytes sedimentation rate ( esr ) , and c - reactive protein ( crp ) .
blood samples were obtained from all subjects into tubes without additives by venous puncture in a fasting state in the morning .
peripheral venous blood was collected into separator vacutainers and allowed to clot for 30 minutes at room temperature .
the test tubes were centrifuged at 3.000  g for 10 minutes , and serum samples were further divided into aliquots and stored at 80c , until assessment . before testing , frozen probes were brought to room temperature , avoiding freezing - unfreezing cycles .
the analysis of serum parameters was based on a quantitative sandwich elisa , according to the manufacturer 's instructions .
igg anti - ccp 3.1 and igg - acl autoantibodies were determined by elisa , using quanta lite-inova diagnostics kits , usa ( autoantibodies seropositivity was defined as a titer > 20 
the investigation of serum igm - rf concentrations was achieved using elisa - aescu germany kits ( positive > 15 
u / l ) and of anti - ra33 antibodies using elisa - kit human , wiesbaden , germany ( positive results > 25  u / ml ) . for hscrp dosage , drg elisa international inc .
serum concentrations of il-13 and il-17 were measured in patients with early , untreated inflammatory arthritis and in control persons , using elisa techniques with invitrogen corporation kits ( camarillo , ca , usa ) .
the values obtained were expressed in pg / ml . in looking for a method of measuring serum cytokine ,
concentrations were taken into account studies that show good stability for samples stored at 70c until dosage .
all the procedures were followed in accordance with the ethical standards of the institutional responsible committees for human studies and with the helsinki declaration of 1975 , as revised in 2008 . for realisation of this study
, we obtained the approval of the committee of ethics and academic and scientific deontology of the university of medicine and pharmacy from craiova number 76/2014 .
patients ' data , management system , and data processing were performed using microsoft excel and data analysis module ; statistical analysis was done using graphpad prism 5 .
the significance of differences between groups was examined with a mann - whitney u test or kruskal - wallis , when multiple comparisons were made .
correlation analysis between the concentration of il-13 and il-17 and the degree of disease activity ( das28 ) , as well as the concentration of some autoantibodies , crp and esr , were conducted with a pearson 's test .
the diagnostic values of studied markers were evaluated using receiver operating characteristic ( roc ) curves analysis .
the performance was expressed as the area under the roc curve ( auc , area under roc curve ) together with 95% confidence interval ( 95% ci ) and p statistics for the difference between calculated auc and auc = 0.5 ( weak discriminative marker ) . cut - off values corresponding to the highest accuracy were determined and for various threshold values investigated at each marker ,
we calculated the sensitivity ( sn ) , specificity ( sp ) , and youden index ( sensitivity + specificity  1 ) .
among the 30 patients , initially diagnosed with era , 80% were female ( sex ratio : 24 female/6 male ) , with age , mean  stdev 55.77  10.87 years . in controls
there was no significant difference in age between the two groups ( table 1 ) . in our study
, we found that both il-13 ( 18.20  pg / ml , 95% ci : 16.4719.92 ) and il-17 ( 17.87  pg / ml , 95% ci : 12.9922.75 ) concentrations in the serum of patients suffering from era were higher than those in the control group ( 4.80  pg / ml , 95% ci : 3.895.71 , p < 0.0001 , and 4.20  pg / ml , 95% ci : 3.365.05 , p < 0.0001 , resp . ) .
we also found differences in serum concentrations of il-13 and il-17 in subgroups of era disease patients with different clinical activity stages .
the concentrations of il-13 in the severe and moderate groups were statistically higher than in the mild and control groups ( p < 0.05 ) .
there were no statistical differences between severe and moderate groups . also , in the case of il-17 serum concentrations increased proportionally with the disease activity of era , the highest concentrations were in patients with severe activity disease ( figure 2 ) .
statistically significant differences were observed between both the moderate and the severe groups and a mild group ( p < 0.05 ) , as well as between the group with moderate disease activity and the control group ( p < 0.001 ) . in the studied cohort of patients , we observe statistically significant differences in the concentrations of crp and the levels of esr between patients with era and the control group ( crp / control group , p < 0.0001 ; esr / control group , p < 0.0001 ) ( table 1 ) . analyzing the relationship between serum levels of crp and esr and different disease activity stages , we observed only statistically significant differences between severe and moderate group ( p < 0.0379 ) ( table 2 ) .
our study showed different profiles of igg anti - ccp and igm - rf concentrations in serum of patients suffering from era in different clinical activity stages .
igg anti - ccp and igm - rf concentrations were increasing along with the disease activity . in both cases , there were statistically significant differences between severe groups and moderate and mild groups ( igg anti - ccp : severe versus moderate group , p = 0.0011 , severe versus mild group , p = 0.0030 ; igm - rf : severe versus moderate group , p = 0.0014 , severe versus mild group , p = 0.0039 ) .
there was a weak , statistically not significant correlation between il-17 and igm - rf ( r = 0.320 , p = 0.085 ) .
there was a significant positive correlation between the concentrations of il-17 and crp ( r = 0.366 , p = 0.047 ) and a significant negative correlation between the concentrations of il-13 and crp ( r = 0.334 , p = 0.041 ) .
disease activity score , das28 , was strongly positively correlated with levels of esr ( r = 0.967 , p  0.001 ) and weakly positively correlated with concentrations of anti - ra33 autoantibodies ( r = 0.404 , p = 0.027 ) .
concentrations of anti - ccp autoantibodies positively correlated fairly well with crp and igm - rf , and concentrations of anti - ra33 positive correlated with levels of esr . comparing
the roc curves for the studied parameters in the patients with era indicated that il-13 has a higher diagnostic utility than il-17 , crp , esr , igm - rf , and anti - ccp as markers of disease activity ( table 4 ) .
roc analysis revealed that il-13 concentration indicated era presence with 100% accuracy using the concentration of 10.73  pg / ml as an optimal cut - off value for discrimination between patients with era and controls ( 95% ci : 0.9621.000 , p < 0.0001 ) .
the likelihood ratios of positive and negative results obtained on the basis of optimal threshold values specific for era were as follows : lr(+ ) = 28.00 and lr( ) = 1.12 with sensitivity and specificity equal to 100 and 100% , respectively ; youden index was 1.00 ( figure 3 ) . in case of il-17 ,
the calculated cut - off value for discrimination between patients with era and controls was 9.40  pg / ml and using this value the diagnostic accuracy of il-17 was 90.2% ( 95% ci : 0.8090.995 , p < 0.0001 ) .
the likelihood ratios of positive and negative results obtained on the basis of optimal threshold values specific for era were as follows : lr(+ ) = 24.27 and lr( ) = 1.14 with sensitivity and specificity equal to 86.67 and 100% , respectively ; youden index was 0.866 . in the studied cohort of patients , crp and esr
have discriminative power towards diagnosis of era ( sensitivities for both crp and esr were found to be lower in comparison to the il-13 ; diagnostic accuracy of crp was 96.9 and 94.8% , resp .
we also noticed that although they have a specificity less than il-13 and crp , autoantibodies igm - rf and anti - ccp have a good tendency to discriminate patients with era from healthy ones ( diagnostic accuracy 98.1 and 94.7% , resp . ) .
chronic inflammatory and autoimmune diseases are the result of an interplay between genetic factors and environmental ones that culminate in the phenotypes of the established disease .
owing to the prevalence and accessibility of joint samples for laboratory investigation , ra has been a suitable model for the study of numerous inflammatory and immune - mediated conditions .
the formulation of a definition for early ra was difficult , but the majority of the rheumatologists use the term  early  for symptoms shorter than three months . there was a tendency to accept the involvement of fewer affected joints [ 9 , 16 ] .
changing from health to established disease in ra is generally clearly understood.early rheumatoid arthritis ( ra ) and very early ra are major targets of research and clinical practice [ 15 , 17 ] .
we found a predominance ( 80% ) of women in patients affected by era , finding which are congruent with the results of other studies having identified a female predominance in ra [ 9 , 15 , 18 ] .
both pro- and anti - inflammatory cytokines were found elevated in ra patients over controls claiming opinion that cytokine networks play critical rolls in the pathogenesis of rheumatoid arthritis [ 4 , 7 , 1928 ] .
the present study reveals that levels of serum il-13 and il-17 cytokines were significantly higher in era patients than in age- and sex - matched healthy persons .
our results are related to the cell - mediated immune response intervention in disease onset .
a broad range of inflammatory processes that are involved in the pathophysiology of rheumatoid arthritis are regulated by cytokines .
the imbalance between pro- and anti - inflammatory cytokine activities favours the induction of autoimmunity , chronic inflammation , and thereby joint damage [ 5 , 27 ] .
some cytokines , such as tumour necrosis factor- ( tnf- ) alpha , il-17 , and ( il)-1 , function by promoting inflammatory responses and by inducing cartilage degradation .
others such as il-4 , il-10 , and il-13 are mainly anti - inflammatory molecules [ 18 , 20 , 25 ] .
even if present in rheumatoid joints , in progressive ra anti - inflammatory cytokine levels are too low to neutralize the deleterious effects of proinflammatory cytokines .
the suppression of both the secretion and action of il-17 by il-13 is of potential clinical importance [ 1922 ] .
il-13 is a protein , secreted by activated t cells , that modulates b cell function in vitro and plays an important part in their proliferation and differentiation ; the high local il-13 levels were observed in patients with ra , correlated with b lymphocyte proliferation .
interleukin 13 induces interleukin-4-independent igg4 and ige synthesis and cd23 expression by human b cells .
some researchers support a role for il-13 as an in vivo antiangiogenic factor and provide a rationale for its use in ra to control pathologic neovascularization .
treatment with th2 cytokines ( il-4 , il-10 , and il-13 ) was tested in many animal models of arthritis based on the th1 bias of t cells , showing considerable promise . in patients suffering from ra , disruptions in self - tolerance lead to abnormalities such as recognition of citrullinated antigens by t and b cells .
the proportion of lymphocyte differentiation in ra is skewed towards the th1 phenotype , to the detriment of the th2 , th17 , and t regulator ( treg ) ones .
imbalances appear in the main cytokine systems including il-1 , tnf , il-6 , il-18 , il-15 , il-33 , il-22 , and il-13 .
however , the destruction of the joint in ra is caused not only by these cytokine imbalances but also by matrix production dysregulation responsible for cartilage damage .
il-17 levels fall after administration of anti - inflammatory cytokines such as il-4 or il-13 .
il-13 levels were significantly higher in patients with early ra ( p < 0.001 ) than in reference group , suggesting the different pathogenic mechanisms involved in joint inflammation .
serum il-13 values increased in ra have been reported in many works [ 20 , 22 , 28 ] . lower interleukin 13 levels were communicated in patients with arthritis by some investigators as barra and contributors   and woods et al . .
assume that the production of rf and antinuclear antibodies by b cells could depend on different cytokines action . in their study , il-13 serum levels correlated with those of rf in ra patients and they suggested that il-13 may be involved in the pathogenesis of autoimmune rheumatic diseases , with a relevant role on rf production .
we found that detection of il-13 in era patients was not affected by rheumatoid factor igm ( r = 0.206 , p = 0.274 ) , a fact revealed by other researchers too .
interleukin 13 inhibits the production of proinflammatory cytokines , chemokines , and hematopoietic growth factors by activated human monocytes .
the increase of biologically active il-13 in ra supports the hypothesis that il-13 regulates immune cell ( including dendritic cell ) activity and indicates how the varied anatomical distribution of cytokines may play a role in the ra disease process .
the differential regulation of circulating il-13 and m - csf levels by tnf antagonists further implies discrete roles in the tnf - cytokine network in ra [ 33 , 34 ] .
.   showed that il-13 was present in 27 out of 28 serum samples from patients with ra , indicating that this cytokine is constantly present in rheumatoid joints .
detected increased levels of the th2 cytokines il-4 and il-13 in synovial samples from early ra patients .
il-13 causes b cell proliferation and differentiation , including ige production , and the expression of certain adhesion molecules on endothelial cells .
all these biological properties of il-13 are shared with il-4 , but in contrast to il-4 , il-13 does not act on t cells . besides the increased values of il-13 , we detected circulating il-17 levels significantly higher in patients with era ( p < 0.001 ) when compared to those in the reference group .
strong correlations of serum il-17a levels with anti - ccp were found by rou et al . .
th17 with decreased circulating levels in era seems to be a marker of anti - ccp seropositivity .
considering the complexity and heterogeneous nature of ra , it is unlikely that only cytokines investigation may provide sufficient discrimination ; predicting the era is better with a combination of biomarkers .
il-17 has several sources : th17 cells , which are a subset of cd4+helper t cells , mast cells , nk cells , and  t cells ; all of them contributing to the pathogenesis of inflammatory arthritis [ 39 , 40 ] . in a recent study comparing individuals before the onset of symptoms ( defined as prepatients ) and after the onset of ra with matched control subjects , kokkonen et al
.   showed that il-17 was present at its highest concentrations in prepatients , and the level had decreased within 7.7 months following the onset of disease .
il-17a was detected at higher levels in early disease compared with late , established disease .
reported il-17a levels significantly higher in synovial fluid ( sf ) and serum from era patients compared to osteoarthritis ( oa ) .
in other previous studies , there were higher serum and sf il-17a levels in ra patients as compared to healthy controls , which suggests that the cytokine is mainly produced locally in the inflamed joint .
other results sustain that despite the significant increases in th17 and il-17 cd4 + t cells in the blood of ra patients , these did not correlate with esr , crp , or das28 , suspecting that the presence of il-17 producing cd4 + t cells in the blood from patients with established ra is of limited use as a biomarker to indicate disease activity . in our study , there were significant correlations ( r = 0.366 , p = 0.047 ) found between il-17 and crp serum levels , but not with esr or disease activity score , supporting partially arguments of these investigators . in another work ,
leipe et al .   demonstrated that th17 cells play an important role in inflammation in human autoimmune arthritides , both at the onset and in established disease .
they claim that the levels of il-17 are connected to the systemic disease activity at both the onset and the progression of ra . taken together
, these data suggest that il-17 may be a key activator of t cell - driven inflammation and thus may contribute to the pathogenesis of ra [ 46 , 47 ] .
it can be asserted that il-17 represents a member of the proinflammatory cytokine family produced by ra synovium and inhibited by some th2 cytokines . in this way , il-17 contributes to the active , proinflammatory pattern characteristic to ra , whose production and function are regulated by il-4 and il-13 .
reduction of synovial inflammation may be protective through a direct effect on il-17 , inducing proinflammatory effects .
thus , il-17 appears to represent a target for treatments of ra [ 48 , 49 ] .
overexpression of il-17 has been shown to be associated with a number of pathological conditions .
since il-17 was found at high levels in the synovial fluid around the affected cartilage in patients with inflammatory arthritis , it is assumed that this determines the direct effect on articular cartilage .
il-17 functions as a direct and potent inducer of matrix breakdown and an inhibitor of matrix synthesis in articular cartilage explants .
it was observed that individuals in whom ra later developed had significantly increased concentrations of several cytokines , closer to the onset of symptoms , and that there exists a relationship between th1 , th2 , treg , and th17-cytokines and the presence of anti - ccp antibodies .
sensitivity , though , was not increased by the combination of anti - ccp antibodies and these cytokines [ 18 , 41 , 51 , 52 ] . because we know the statistical limits of the present study , the relatively small number of patients , we propose in the future longitudinal studies with regular serum analysis to determine more precise roles of il-17 , il-13 , and autoantibodies in ra pathogenesis .
with il-13 and il-17 serum concentrations increasing proportionally with the disease activity of era , the highest concentrations were in patients with severe activity disease . based on the results of this study , we can conclude that the presence of higher il-13 , il-17 , igm - fr , and anti - ccp serum levels in patients , compared to those of controls , confirms that these markers , found with high specificity , might be involved in the pathogenesis of era . il-13 and il-17 might be of better usefulness in the prediction of era activity status than igm - rf and anti - ccp .
investigation of the association between cytokine profile and autoantibodies status may lead to prognostic and treatment decisions in era patients .
the basis for the new therapies in patients with ra is represented by inhibiting the action of proinflammatory cytokines by using specific cytokine inhibitors or anti - inflammatory cytokines .
the combination of il-17 and anti - ccp autoantibodies may have the potential as biomarkers in early ra , especially for their clinical utility .
 
 aims . in the present study , we aimed to assess the concentrations of il-13 and il-17 in serum of patients with early rheumatoid arthritis ( era ) , the investigation of correlation between the concentrations of these cytokines and disease activity score , and the concentration of some autoantibodies and the evaluation of the utility of il-13 and -17 concentration measurements as markers of disease activity . 
 materials and methods . 
 serum samples were collected from 30 patients and from 28 controls and analysed parameters . 
 results . 
 the serum concentrations of il-13 , il-17 , anti - ccp , and igm - rf were statistically significantly higher in patients with era , compared to the controls . 
 il-13 concentrations in the severe and moderate groups with era were statistically higher than in the mild and control groups . 
 also , in the case of il-17 , serum concentrations increased proportionally with the disease activity of era . 
 we observe that concentrations of il-13 and -17 did not correlate with autoantibodies . 
 il-17 concentration significantly positively correlated with crp , while il-13 concentration significantly negatively correlated with crp . 
 disease activity score , das28 , was strongly positively correlated with levels of esr and weakly positively correlated with concentrations of anti - ra33 autoantibodies . 
 il-13 has a higher diagnostic utility than il-17 , crp , esr , igm - rf , and anti - ccp as markers of disease activity . 
 conclusions . 
 the presence of higher il-13 and il-17 serum levels in patients , compared with those of controls , confirms that these markers , found with high specificity , might be involved in the pathogenesis of era . il-13 and il-17 might be of better usefulness in the prediction of era activity status than igm - rf and anti - ccp . 