Basic And Clinical Study On The Serum Cytokines And Magnetic Resonance Imaging In Thyroid Associated Ophthalmopathy

BACKGROUNDThyroid associated ophthalmopathy(TAO) is considered to be a common organ specific autoimmune inflammatory disorder that is closely associated with thyroid disease, especially affecting at least 80% of Graves' disease(GD) patients, so we call it Graves ophthalmopathy (GO). The most common clinical features of Graves' ophthalmopathy are proptosis,upper eyelid retraction, edema, and erythema of the periorbital tissues and conjunctivae, and visual field defects or vision loss,affect life of TAO patients.Although genetic determinants of Graves' ophthalmopathy remain poorly understood. it is believed that autoimmune T cells, B cell activation is the inevitable link in the pathogenesis of autoimmune disease. Similar with the other autoimmune diseases.TAO also has many kinds of cytokiens of imbalance and abnormalities in the body.T lymphocytes trigger a cascade of events, including secretion of cytokines. These cytokines stimulate the proliferation of orbital fibroblasts, expansion of adipose tissue, and secretion of hydrophilic glycosaminoglycans from fibroblasts. So as to increase orbital content can explain many manifestations of Graves' ophthalmopathy. We believed that at least three kind of CD4+ Th cell involved TAO:Thl,Th2 and ThO. Thl cell releases cytokines IL-2,IFN-γ,TNF-α,and IL-12 to mainly mediated cell immune.Th2 cell is connected with B cell differentiation and antibody production, bias to produce cytokines IL-4, IL-5, IL-6, IL-10, etc.Th17 cell is formed by natural T cell,and has mechanism of independent differentiation and regulatory. Nanba' report indicated that Thl7 cells also participated in autoimmune disease development and had certain relevance with disease severity.In short, in the condition of normal physiologics,Thl,Th2 and Thl7cell regulation make our body's immune response state in a nice and complex balance. Once the balance is broken, our body may occur in autoimmune disease. But how those cells and related cytokines to work in TAO were not fully understood.Liquid chip is a new chip technology platform which based on the United States xMAP technology Luminex company. Applied in a variety of cytokines detection, can judgment immunological function overall, and understanding the molecular level of immune regulation mechanism.The natural course of TAO was characterized by an active (inflammatory) phase in the initial stage, subsequently followed by an inactive (fibrotic) phase in the late stage. One of the goal of medical treatment is to inactivate the autoimmune process. The assessment of disease activity is absolutely essential in predicting the outcome of medical management in TAO, because medical treatments are generally effective in patients with active TAO, but not in those with inactive disease.Magnetic Resonance Imaging (MRI) is a non-invasive, no-radiation video diagnostic technology, with good organization and spatial resolution, it not only can clearly show the orbital tissue in anatomical structure, but also can indirect reflect inflammation and swelling of the tissue, and can be quantitative analysis, therefore, it is an important tool in clinical diagnosis of TAO.T2 value is the time as horizontal magnetized relaxation attenuation to the 37% of maximum signal intensity,it can be quantitative measured by MRI T2 Mapping. Foreign studies showed joint MRI orbital SIR and orbital T2 value,not only can find tissue edema,but also can quantitativly analyze inflammation of tissue, thus predict immunosuppressive therapy.In Mourits' report,approximately half of patients who had 1 or 2 points in CAS showed significant response to immunosuppressive treatment.This result indicated that CAS alone was inadequate for the evaluation of TAO.Therefore,joint other modalities such as orbital MR imaging can improve the diagnostic sensitivity. And such research report about indexes of MRI had so fewer in domestic.To gain insight into the immune mechanisms responsible for TAO in further, in our study, we use bead-based multiplex sandwich immunoassay(Luminex) to detech serum Th1-,Th2-,Th17 derived cytokines and chemotactic fators in TAO patients, And through quantitative measurement MRI orbital T2 value of EOM and lacrimal gland and dynamic observation orbital SIR change, further discussed MRI in the judgment of active TAO.ChapterⅠserum cytokines in the the pathogenesis of Thyroid associated ophthalmopathyObjectiveTO explore serum Th1-,Th2- and Th17-derived cytokines in the pathogenesis of TAO using Luminex.Materials and methods1.Subjects45 TAOcases with stable thyroid function and not receive high dose glucocorticoid pulse and other immunosuppressive agents treatment Nearly three months were consecutivly collected in Dep.Endocrinology and Metabolism.25 patients with active TAO(CAS>3),20 patients with inactive TAO(CAS<3).10 patients with Graves' disease without ophthalmopathy(GD),10 healthy volunteer were collected meanwhile.serum Th1-,Th2- and Th17-derived cytokines were determined usig Liquichip in above four groups.2. Statistical analysisStatistical analyses were conducted with SPSS 13.0 for windows. All data are presented as means±SD. Differences between groups were analyzed using One-way analysis of variance (ANOVA), and multiple comparisons were analyzed by LSD method when P values less than 0.05. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett T3 method when P values less than 0.05. Statistical significance was accepted at a value of P<0.05.The correlation between serum cytokines and TRAb were analyzed using Spearman relation.Results1.Results of Th1 derived cytokines:IFN-γ:The serum concentration of IFN-γin active TAO,inactive TAO were higher than that in the controls. There were significant differences in serum concentration among these three groups (P=0.000 P=0.000), and there was statistic difference between active TAO and inactive TAO(P=0.000), but there was no statistic difference between active TAO and GD group (P>0.05);TNF-a:The serum concentration of TNF-a in active TAO,inactive TAO and GD were higher than that in the controls. There were significant differences in serum concentration among these four groups(P=0.000 P=0.015 P=0.043), and there was statistic difference between active TAO and inactive TAO(P=0.043); IL-1a:The serum concentration of IL-1a in active TAO-. inactive TAO,GD were higher than that in the controls. There were significant differences in serum concentration among these four groups (P=0.001 P=0.000 P=0.000), but there was no statistic difference among experimental groups (P>0.05)IL-1ra:There was no statistic difference among serum concentration of IL-1ra in four groups (F=0.894 P=0.450).IL-2:The serum concentration of IL-2 in GD was higher than that in the controls. There were significant differences in serum concentration between them (P=0.000), but the serum concentration of IL-2 in TAO and control had no significantly, and compared with GD group, the serum concentration of IL-2 in active TAO and inactive TAO group decreased significantly(P=0.000 P=0.000).Results of Th2 derived cytokines:IL-6:The serum concentration of IL-6 inactive TAO,inactive TAO,GD were higher than that in the controls. There were significant differences in serum concentration among these four groups(P=0.000 P=0.000 P=0.000), and the serum concentration of IL-6 inactive TAO,inactive TAO were lower than that of GD group(P=0.000 P=0.000);IL-4:The serum concentration of IL-4 in active TAO,inactive TAO and GD were higher than that in the controls. There was significant differences in serum concentration among these four groups(P=0.000 P=0.001 P=0.000), and compared with GD and inactive TAO, the serum concentration of IL-4 in active TAO significantly decrease, there was statistic difference among those three groups (P P=0.000 P=0.000)Results of Th17 derived and other related chemotactic factors:IL-17:The serum concentration of IL-17 in active TAO,inactive TAO and GD were higher than that in the controls. There was significant differences in serum concentration among these three groups(P=0.000 P=0.000 P=0.028), but there was no statistically significant differences among experimental groups (P>0.05).IP-10:Compared with the serum concentration of IP-10 in conontrols, there were significant differences in serum concentration among these four groups(P=0.003 P=0.034 P=0.000), And in TAO groups, IP-10 levels decreased compared with GD group (P=0.006 P=0.003),but there was no statistic difference between active TAO and inactive TAO (P>0.05)RANTES:showed no statistic difference among all groups(F=0.212 P=0.887).2.Correlation results among serum cytokines and TRAb in TAO groupsThe serum of IFN-γ,TNF-a,IL-1a,IL-2,IL-4,IL-6 in TAO had correlation with TRAb(r=0.409,0.309,0.310,-0.352,-0.340,0.332; P=0.005,0.039, 0.038,0.018,0.022,0.026),but other serum cytokines and chemotactic factors had no significant correlation with TRAb(P>0.05).Conclusions1. The serum concentration of Th1-,Th2- and Th17-derived cytokines in GD,TAO was higher/lower than those in the controls, which showed that Th1-,Th2- and Th17-derived cytokines played important roles in the pathogenesis of two diseases;2. Some Serum Th1-,Th17-derived cytokines incresed in active TAO,and some Th2 derived cytokines increased in inactive TAO,a disturbed balance between serum Thl-,Th2- and Th17-derived cytokines may decide the reactiveness of TAO in some degree. Chapter II The role of T2 values and signal intensity ratio on MR images in the asssessmemt of active thyroid associated ophthalmopathyObjective1.Quantitative measure extraocular muscles and lacrimal gland T2 values on magnetic resonance imaging, analysis the correlation between orbital T2 relaxation time and clinical activity score(CAS),and to find out an objective and quantitative method to assess active TAO.2. Dynamic viewing the change of extraocular muscles signal intensity ratio(SIR) on MR images before and after radiotherapy, and comparing with our previous research on TAO, to further clarify the role of orbital SIR in the assessment of active TAO.Materials and methods1.Subjects1.1 T2 value measurement:the same as chapterⅠ. Except for fewer people refuse to MRI scan, consecutively collected 43cases TAO(23 active TAO and 20 inactive TAO),8cases GD and 8 healthy volunteer,using MRI T2 Mapping to quantitative measure extraocular muscles and lacrimal gland maximal and average T2 values respectively.1.2 Dynamic viewing the change of orbital SIR:26cases TAO after radiotherapy were consecutivly collected from January 2008 to October 2009 in Dep. Endocrinology and Metabolism.12 males and 14 females, average age was (37.85±10.74)years.The duration after radiotherapy for TAO was(15.62±2.33)months.TAO was diagnosed according to Bartleys. Cases inclusin criteria:①received radiotherapy during January 2008 to October 2009;②The duration after radiotherapy for TAO 12 to 18 months,and not receive further treatment during follow-up,except for eyedrops;③have complete MRI datas(FSE T2WI+fat inhibition sequence), now CAS<3,and with stable thyroid function;④with no other autoimmune diseases, no diabetes and hypertension,and now Liver and kidney function were normal;⑤had complete clinical datas;⑥patiens want to receive MRI scan voluntarily,and had not MRI contraindications.All the patients receive MRI scan again.2. MRI examination protocol.All the patients were examined with GE SIGNA EXCITE 3.0T MRI scanner,measureing extraocular muscles T2 values using MRI T2-Mapping scan.SIR was measured by coronal fast spin echo T2WI plus to fat inhibition sequence scan,and proptosis was measured by transverse TIWI scan.The agreement of MRI were assessed by two doctors of Medical Imaging Department.3. Clinical indexesAll patients received CAS assessment,protosis measurement and TRAb detection again.Above indexes were performed by Two designated specialized personnel,and come to an agreement.4. Statistical analysisStatistical analyses were conducted with SPSS 13.0 for windows. All data are presented as means±SD. Differences between groups were analyzed using One-way analysis of variance (ANOVA), and multiple comparisons were analyzed by LSD method when P values less than 0.05. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett T3 method when P values less than 0.05. The relation among T2 values,CAS and TRAb were analyzed using Spearman relation.The relation among CAS,SIR,proptosis and TRAb before and after radiotherapy were analyzed using Spearman relation respectively. The change of CAS,SIR,proptosis and TRAb before and after radiotherapy using paired sample t-test. Statistical significance was accepted at a value of P< 0.05.Results1. Results of MRI-T2 values and the correlation among them and CAS> TRAb1.1 The results of extraocular muscles T2 values on MR images in different groupsextraocular muscles maximal T2 values and average T2 values in active TAO,inactive TAO and GD were higher than that in the controls, there were significant differences in serum concentration among these groups(the former P=0.000,0.004,0.000, the latter P=0.000,0.002,0.003), extraocular muscles maximal T2 value and average T2 value in active TAO were significantly higher than that of GD and inactive TAO group(the formerP=0.007,0.012; the latter P=0.000,0.002).1.2 The results of lacrimal gland T2 values on MR images in different groupslacrimal gland maximal T2 values in active TAO,inactive TAO and GD were higher than that in the controls, there were significant differences in serum concentration among these groups(P=0.000,0.005,0.021), compared with control group, the average T2 values in TAO increased significantly(P=0.000, P=0.046), lacrimal gland maximal T2 value and average T2 value in active TAO were significantly higher than that of GD and inactive TAO group(the formerP=0.034, 0.019; the latter P=0.045,0.008).1.3 Correlation results among extraocular muscles and lacrimal gland T2 values,CAS and TRAbThere were positive correlation between extraocular muscles maximal T2 value, average T2 value and CAS(r=0.493,0.507 P=0.001),and so were lacrimal gland(r=0.494,0.521 P=0.001,0.000). There were positive correlation between orbital extraocular muscles maximal T2 value, average T2 value and TRAb(r=0.438,0.414 P=0.003,0.006), lacrimal gland maximal T2 value had positive correlation with TRAb too(r=0.359 P=0.018).2. Longitudinal observe the MRI-SIR before and after radiotherapy and the correlation among them and other clinical indexes2.1 The change of each indexed before and after radiotherapySIR was (1.85±0.20) before radiotherapy, and SIR was(1.51±0.17),SIR after radiotherapy significantly decrease compared it with before treatment(t=7.14, P< 0.001),so did CAS,TRAb and proptosis(all P<0.001).2.2 Correlation results among SIR and CAS,TRAb,proptosis before radiotherapyThere were positive correlation among SIR and CAS,TRAb(the former r=0.430 P=0.0285; the latter r=0.429 P=0.0288),but there was no Significant correlation between SIR and protosis(P=0.0714).2.3 Correlation results among SIR and CAS,TRAb,proptosis after radiotherapyThere were positive correlation among SIR and CAS(r=0.475 P=0.014),TRAb(r=0.424 P=0.031),proptosis(r=0.515 P=0.007).2.4 Correlation results between the change of SIR and the change of CASThere was positive correlation between SIR D-value and CAS D-value before and after radiotherapy(r=0.473 P=0.015).Conclusion1.MRI T2 value of extraocular muscles lacrimal gland could be to assess active TAO,and orbital T2 value increased in active TAO,there were positive correlation among T2 value of extraocular muscles lacrimal gland and CAS,TRAb.2.Extraocular muscles SIR significantly decrease after radiotherapy, and the change of SIR had positive correlation with the change of CAS,so we say,SIR not only can assess active TAO,predit the therapeutic effect of radiotherapy of TAO,but also guild to dynamic view the change of TAO.