The Study Of IL-1β Stimulates Expression And Production Of RANTES In Human Retro-orbital Fibroblasts

Objective: Graves ophthalmopathy (GO) is generally considered to be an chronic autoimmune disorder that is closely associated with autoimmune thyroid disease and usually involves all orbital tissue compartments.But its pathogenesis remains unclear.Given our current state of knowledge, the following working scheme for the pathogenesis of GO can be proposed: On the background of a permissive immunogenentic milieu, circulating T cells in patient with GO, directed against certain antigens on thyroid follicular cells, recognize antigenic epitopes that are shared by tissues contained in the orbital space.T cells recruitment into the orbital tissues are facilitated by certain chemokines and cytokines. T cells and macrophages populating the orbital space are known to synthesize and release a number of cytokines into the surrounding tissue. Retro orbital fibroblasts (RF) can be stimulated to proliferate, synthesize glycosaminoglycan (GAG) and express immunomodulatory molecules by these cytokines. Which may contribute to the edema, denature of orbital tissues and extra ocular muscle and then lead to proptosis diplopia periorbital edema and inflammation. Thus, the exploration of the mechanism of expression of chemokines and infiltration of T cells into orbital tissues may have important implication in the pathogenesis of GO. RANTES is a member of CC-type chomekine.The main role of RANTES is to recruit monocytes and T cells to inflammatory sites There are several reports demonstratingthat the presence of RANTES and it's receptors in the thyroid tissues of patients with GD.Thus RANTES may also be associated with the pathogenesis of GO. Received appropraite stimulation , b100d plates and fibroblasts can become another main sources for RANTES. IL-1β can up-regulates expression of chemokines in various tissues and cell types. It has also been demonstrated that IL-1β is highly expressed in the eye muscle of patients with GO and play an important role in the pathogenesis of GO. Thus, it is important to investigate factors such as IL-1β regulating the expression of RANTES in RF. Since the binding sites of NF-kB are present on the promotors of CC-chemokines (MIP-1a MIP-1β RANTES), we hypothesize that NF-kB is prerequisite for IL-1β-mediated up-regulation of RANTES in RF.The objects of this study include that: (1) To study the effect of IL-1β on RANTES expression and production in cultured RF.(2) To determine the chemoattractant activity of RANTES produced by RF; (3) To investigate the possible molecular mechanism of IL-1β-induced expression of RANTES.Materials and Methods : RF were obtained by digesting the retro orbital connective tissue with collagenase from subjects with GO (n=4) and strabismus as controls (n=4). RF in (2-10) were cultured in 24-well cuture plate and incubated with IL-1β (0, 0.04, 0.4, 8, 10ng/ml), CAPE (50ug/ml) IL-1β (10ng/ml) +CAPE (50ug/ml) . Protein and mRNA levels of RANTES were determined by ELISA and RT-PCR. Migration of PBL to IL-1β stimulated RF medium was evaluated by chemotaxis assay using 96-well microplate chemo TX system.3.25 mm diameter, 5um pore polycarbonate membranes were placed over the wells of a corresponding 96-well tissue culture plate.The bottom wells of the chamber were divided into 4 groups according to RF cultures added in : (1) From RF culture medium lacking experimental agents.(2) From RF conditioned culture medium containing IL-1β(10ng/ml).(3) Pre-incubation of RF conditioned medium containing IL-1β(10ng/ml) with mouse-anti human RANTES neutralizing antibody.(4) Pre-incubation of RF medium lacking experimental agents with mouse-anti human RANTES neutralizing antibody.PBL (3 X 106/ml) in 50ul of culture were added to the upper side of the membrane and the plate was incubated at 37C for 3h.The cells which migrated to the lower wells wereenumerated by light microscopy. Results1. At the beginning of separation, cells exist in single globular shape. RF exhibit fusiform and angular shapes with multiple dendritic processes and proliferate as a monolayer ab...