Significance Of Tear Cytokines In Measuring Disease Activity In Thyroid Associated Ophthalmopathy

BACKGROUNDThyroid associated ophthalmopathy (TAO) is considered to be a common organ specific autoimmune inflammatory disorder that is closely associated with Graves disease, so we call it Graves ophthalmopathy (GO). Its Ocular clinical manifestations are unique. Mild TAO always present as eyelid retraction and discomfort of eyes, while in severe cases, proptosis, diplopia, and even loss of vision may happen. There is no satisfactory treatment until now.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. Graves’ophthalmopathy is probably initiated by autoreactive T lymphocytes reacting with one or more antigens shared by the thyroid and orbit; after reaching the orbit and recognizing the shared antigen (or antigens), 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. The resulting increase in orbital content explains many manifestations of Graves’ophthalmopathyT 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. The thyrotropin receptor is the primary autoantigen in Graves’ophthalmopathy. Obital fibroblasts, lymphocytes and cytokines play a key role in the occurrence and development of TAO.The role which lacrimal has played in the pathogenesis of TAO still is not known. Imaging study of TAO showed enlargement of the lacrimal gland in certain cases. Pathologic examinations of lacrimal glands in active TAO showed increased numbers of lymphocytes and interstitial edema. Eckstein founded that ocular surface damage in TAO correlates with reduced tear production, and acinar cells from lacrimal glands were positive for thyotropin receptor (TSHR), which is considered a target of autoimmunity shared by both thyroid and extrathyroidal sites in TAO.The reaserchers puted forward a hypothesis that the acinar cells of lacrimal maybe one of target cells of TAO.Clinically, the symptoms of TAO patients with ocular surface are prominent, including conjunctival congestion and edema, tears or reduced tear secretion, dryness and sandiness, causing the attention of clinicians and scholars. The most likely mechanism is the increased evaporation of the tear film inTAO patients, resulting in evaporative dry eye. This is the result of increase in volume of the orbital contents that push the globe anteriorly and increase the palpebral fissure width. Proptosis secondarily causes evaporative loss of the tear film and increases the tear film osmolarity. Recent evidence demonstrated additional possible mechanisms that may contribute to the pathogenesis of dry-eye symptoms. The protective for the ocular surface of lacrimal gland and normal immune function rely on the secretion of tears. Tear Contains complex cytokine composition, lacrimal gland maintain the stability of the ocular surface and participate in corneal epithelial wound healing through through secreting cytokines.In addition, cytokines can regulate a variety of cellular functions, its concentration can cause a range of inflammatory response, can also be used as markers of disease recovery signal and the dry eye and other ocular surface diseases.Lacrimal glands were positive for TSHR as same as obital fibroblasts, the pathologic change of lacriaml in TAO is similar with obital issue, add to the symptoms of ocular surfac, so we speculate that the cytokine network in lacrimal is unbalance as in orbital. We wonder that whether the expression of tears cytokines is related to TAO? Whether Cytokine levels can reflect indirectly the lacrimal gland inflammation and disease activity of TAO? There is little this kind of studies at home and abroad, the expression tear cytokine is not entirely clear now. The study of tear cytokine profile helps to further explore the role of the lacrimal gland in the pathogenesis of thyroid associated ophthalmopathy and provide possible new targets for the treatment of TAO. Comparison of cytokine concentrations in different phases of TAO helps seeking possible indicators of judgment of active TAO and to provide a reliable basis for clinical treatment.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.To further explore the role of lacrimal gland and tear cytokines in the pathogenesis of TAO and seek possible indicators for judgment of active TAO, we use bead-based multiplex sandwich immunoassay (Luminex) to detect tear IL-6, IL-10, TNF-α, IFN-γ, IL-17, IL-8and IP-10level in TAO patients, the change of tear cytokines levels before and after orbital radiation therapy were also researched.Chapter I Analysis of tear cytokine profile in active and inactive TAOObjectiveTo further explore the role of lacrimal gland and tear cytokines in the pathogenesis of TAO and seek possible indicators of judgment of active TAO by detecting seven kinds of tear cytokines in TAO using Luminex.Materials and methods1. Subjects47TAOcases 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.27patients with active TAO(CAS>3),20patients with inactive TAO(CAS<3).10healthy volunteer were collected meanwhile.Tea IL-6, IL-10, TNF-α, IFN-γ, IL-17, IL-8and IP-10were determined usig Liquichip in above three groups.2. Statistical analysisStatistical analyses were conducted with SPSS13.0for windows. All data are presented as means±SD or median. When the data fit the normal distributionand, differences between groups were analyzed using One-way analysis of variance (ANOVA), multiple comparisons were analyzed by LSD method when P values less than0.05. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett T3method when P values less than0.05. When the data do not fit the normal distributionand, differences between groups were analyzed using non-parametric tests. The concentration of tear cytokines in the three groups were compared using the Kruskal-Wallis H test, multiple comparisons were analyzed using the Wilcoxon signed rank test, Statistical significance was accepted at a value of P<0.017..The correlation between tear cytokines and clinical activity score(CAS) were analyzed using Spearman relation.Statistical significance was accepted at a value of P<0.05Results1. Tear cytokine profile in TAO and control group. IL-6, IL-8and IP10were detected in all57Cases (detection rate100%),The concentration range of IL-6was from0.32pg/ml tol583.15pg/ml. The concentration range of IL-8was from58.26pg/ml to19404.08pg/ml,the range of IP10was from6098.18pg/ml to24034pg/ml.TNF-a and IL-10were detected in54cases (detection rate94.7%), the concentration range of TNF-α were from0.25pg/ml to121.23pg/ml, the concentration range of IL-1were from0.33pg/ml to108.82pg/ml. IL-17is only detected in1case (detection rate1.8%), IFN-γ were not detected in all samples.2. Comparision of tear cytokine concentrationsbetween TAO and the cintrol groupCompared with the control group, tear IL-6, IL-10, IL-8and IP-10levels of TAO patients were significantly increased, the difference was statistically significant (P<0.001P=0.046P=0.002P=0.026), while there was no significant difference of tear TNF-α between TAO and control group (P=0.664).3. Tear cytokine concentrations between the different groupsIL-6:Compared to the control group, the concentrations of tear IL-6of active TAO group and the inactive TAO group were significantly increased, the difference was statistically significant.(P<0.001P=0.003) between active TAO group and the inactive TAO group, the difference was statistically significant, tear concentrations of IL-6of active TAO group were significantly higher than the inactive TAO group (P=0.001<0.017).IL-10:compared to the control group, the concentrations of tear IL-10of active TAO group was significantly increased, the difference was statistically significant (P<0.001); the tear IL-10concentration in active TAO was significantly higher than inactive TAO, the difference was statistically significant (P=0.009); difference between the active TAO group and control group was not statistical significant (P=0.705)TNF-a:Compared to the control group, the concentrations of tear TNF-a the active TAO group and inactive TAO group was not significantly different (P=0.353P=.555), the difference between active TAO group and inactive TAO group was statisticantly (P=0.006).IL-8:compared to the control group, the tear IL-8concentration of active TAO was significantly increased, the difference was statistically significant (P=0.000); the difference between the active group and the inactive group was not statistically significant (P=0.017).the difference between the inactiveTAO group and control group was no significant (P=0.044).IP-10:compared to the control group, the concentrations of tear IP10of active TAO group was significantly increased, the difference was statistically significant (P=0.004); the tear IP10concentration in active TAO was significantly higher than inactive TAO, the difference was statistically significant (P=0.004); difference between the active TAO group and control group was not statistical significant (P=0.307)4. Correlation results among tear cytokines and CAS and TRAb in TAO groupsThe concentrations of tear IL-6、IL-10、TNF-α、IL-8、IP10in TAO had correlation withCAS(r=0.565,0.415,0.453,0.385,0.469; P<0.001,0.005,0.002, Tear IL-6、TNF-α、IL-8、IP10concentrations in TAO had correlation withTRAb (r=0.477,0.325,0.314,0.365; P=0.002,0.032,0.032,0.012),there was no correlation between TRAb and tear IL-10.Conclusions1. The concentration of tear cytokines IL-6, IL-10, IL-8and IP10in TAO were increased sigificantly than healthy peoples.2. The expression of tear cytokines in different phase of TAO were siginificantly different, tear IL-6, IL-10and IP10level in active TAO was significantly higher than inactive TAO.and was positively correlated with CAS. To a certain extent, tear IL-6, IL-10and IP10concentration could be used as indicators for judgment of activity of TAO. Chapter II Changes of tear cytokines before and after orbital radiotherapy in thyroid associated ophthalmopathyObjectiveLongitudinal observation of the seven kinds of tear cytokines levels thyroid-associated ophthalmopathy who had received orbital radiotherapyMaterials and methods1. Subjects1.14cases of active TAO group in firdt chapter who had Received orbital radiation therapy and had integrity follow-up data after treatment (2males and12females), average age (39.00±11.17years). to detect the tear IL-6, IL-10, TNF-α and IFN-γ, IL-8, IP-10and IL-17levels using liquid chip technique.2. Orbital radiation treatmentVARIAN2100C linear acceleratorthe(manufacturefd from United States).Fractionated doses of radiation2Gy,total dose of20Gy(10times/2weeks), in the simulator under the positioning, size of radiation field is generally4cm×5cm, deflection angle rack3°～5°in order to contralateral to avoid crystal,treatment lasted for2weeks. Patients should receive oral prednisone30mg1/day, tapering to discontinuation at the end of treatment.3. Clinical indexesAll patients received CAS assessment, NOSPECS classification, protosis and palpebral fissure 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 SPSS13.0for windows. All data are presented as means±SD or median. The change of CAS, palpebral fissure width, proptosis and TRAb before and after radiotherapy using paired sample t-test. tear cytokine concentrations before and after treatment were compared using non-parametric test for two related samples.Statistical significance was accepted at a value of P<0.05.Results1. The comparison of clinical indicators before and after radiation therapy after radiation therapyCAS scores, palpebral fissure width and exophthalmos degree were significantly decreased after therapy, the difference were statistically significant (P<0.001P=0.001P=0.005P=0.009)2. Change of tear cytokines before and after treatment Tear IL-6concentration was significantly decreased after treatment (P=0.041). there was no statistic difference of Other cytokines including IL-10, TNF-α, IL-8and IP10before and after therapy(P=0.950P=0.074P=0.096P=0.510).Conclusion1. Tear IL-6concentrations decreased significantly after orbital radiotherapy, the possible mechanisms maybe the ray of radiotherapy reduce the infiltration of lymphocytes and synthesis and secretion of IL-6in lacrimal gland, And then reduce tear IL-6concentration.2. The orbital radiation therapy is effctive in the treatment of active moderate to severe TAO.Clinical activity score, the exophthalmos and palpebral fissure width decreased significantly after treatment.