A study on the establishment of co-culture system of peripheral blood mononuclear cells with orbital fibroblasts and the secretion of IL-6 and IL-17A induced by phytagglutinin in thyroid associated ophthalmopathy

Background

The pathogenic mechanism of thyroid associated ophthalmopathy (TAO) is still unclear, which is considered to be an autoimmune disease.It is confirmed that interleukin-17A (IL-17A) plays an important role in the occurrence and development of many autoimmune diseases.It is unclear that whether IL-17A participates in the pathogenesis of TAO.

Objective

This study was to explore whether IL-17A secreted by co-culture system of peripheral blood mononuclear cells (PBMCs) and orbital fibroblasts (OFs) participates in the pathogenesis of TAO and its possible mechanism.

Methods

Periphery blood and orbital connective tissue were obtained from 12 patients with TAO and 8 patients who received prosthesis implantation for eyeball atrophy in Xiangya Hospital during April to December 2014.PBMCs were isolated by density gradient centrifugation, and OFs were cultured by explant culture method.The purity of T leukomonocyte in PBMCs was tested by flow cytometry, and OFs were identified by Giemsa staining and immunochemistry.OFs and PMBCs were incubated into 96-well plate in a 1∶20 proportion to establish co-culture system.Different concentrations of phytagglutinin (PHA) (0, 1.0, 2.5, 5.0, 10.0 μg/ml) was added for 72 hours, and IL-6, IL-17A levels in the co-culture system supernatant and IL-17A receptor (IL-17RA) of the total cell membranes in the co-culture system were assayed by ELISA.The differences of IL-6, IL-17A, IL-17RA levels in co-culture system were compared between the TAO group and control group.

Results

The mean purity of T leukomonocyte in PBMCs was (81.10±0.21)% in the TAO group and (80.05±0.38)% in the control group respectively, with no significant difference between them(t=0.923, P>0.05). Cultured OFs showed the positive response for Vimentin expression and Giemsa staining.After stimulated by 1.0 μg/ml PHA, the proliferation of both PBMCs and OFs were increased in the co-culture system.Apoptosis exist in PBMCs and the number of OFs decreased when PHA was higher than 1.0 μg/ml.The growth of PBMCs and OFs was faster in the TAO group than that in the control group in the same concentration of PHA.The contents of IL-6, IL-17A and IL-17RA in co-culture system were significantly different among various concentrations of PHA subgroups (IL-6: F_group=12.561, P=0.000; F_{concentration}=23.356, P=0.001.IL-17A: F_group= 12.037, P=0.000; F_{concentration}=19.206, P=0.000.IL-17RA: F_group=16.216, P=0.000; F_{concentration}=4.627, P=0.018). The production of IL-6, IL-17A and IL-17RA reached peak in both TAO group and the control group after 1.0 μg/ml PHA stimulated.However, the concentrations of IL-6, IL-17A and IL-17RA reduced with the increase of PHA concentration.The concentrations of IL-6, IL-17A and IL-17RA in co-culture system were significantly higher in the TAO group than those in the control group under the stimulation of the same concentration of PHA (all at P<0.05).

Conclusions

The co-culture system of PBMCs and OFs stimulated with PHA can be the imitation of TAO pathogenesis in vitro, and PHA can amplify its immune reaction to imitate TAO pathogenic processes intuitively.The IL-6, IL-17A and IL-17RA secreted by PBMCs and induced by PHA are increased in TAO patients, implying that IL-17A participates in the pathogenesis of TAO through magnifying cellular immune response and inflammatory reaction.

Cell culture; Thyroid-associated ophthalmopathy; Peripheral blood mononuclear cells; Orbital fibroblasts; T lymphocytes; Interleukin-6; Interleukin-17A; Human