The Study Of Mechanisms Of Inflammatory Cytokines Production In The Obstinate Ocular Fundus Diseases

Part I Proteasome inhibitor MG-132up-regulates IL-6expression in RPE cells through the activation of P38MAPKsPurposeAs far as we know, during the development of age-related maculardegeneration (AMD), the activity of proteasome in retinal pigment epithelium cells(RPE) gradually decreases. And a lot of research has shown that age-relatedmacular degeneration is closely related to inflammation and autoimmune. Moreover,there are many cytokines (CKs) involved in the course of inflammation. In thisstudy, we are going to investigate how the decrease of proteasome activity affect theproduction of interleukin-6(IL-6) in human retinal pigment epithelium cells(ARPE-19).MethodsCultured ARPE-19in Dulbecco's modified Eagle's medium (DMEM),containing10%fetal bovine serum (FBS),100U/ml penicillin G and100μg/mlstreptomycin, was treated with or without MG-132, a proteasome inhibitor, and the levels of IL-6mRNA (Messenger Ribonucleic Acid) in RPE at1h,4h,8h and IL-6protein in the culture medium at2h,4h,6h,8h,10h,12h were measured byReal-time polymerase chain reaction (Real-time PCR) and enzyme-linkedimmuno-sorbent assay (ELISA). The protein levels of MCP-1(mono-cytechemo-attractant protein-1) in the culture medium at2h,4h,6h,8h,10h,12h wasalso measured by ELISA. Then we tested which of cell signal pathways regulatingthe production of IL-6were activated when we added MG132into the medium byelectrophoretic mobility shift assays (EMSA) and Western-blot. After that, we putthe inhibitors of these activated cell signal pathways into the medium individuallyto see which inhibitor can counteract the effect of up-regulating the levels of IL-6inthe culture medium of RPE.ResultsMG132decreased the secretion of MCP-1in the culture medium of RPE, but itincreased the expression of IL-6mRNA in RPE and IL-6protein level in the culturemedium of RPE. MG-132treatment was also found to enhance the level ofphosphorylated p38Mitogen-Activated Protein Kinases (MAPKs) and c-JunN-terminal Kinase (JNK) by western-blotting. More importantly, the effect of MG132on up-regulating the levels of IL-6was counteracted by the pretreatment of the cellswith SB203580, an inhibitor of P38MAP kinases. But the JNK inhibitor, SP600125,can not counteract the effect of up-regulating the levels of IL-6by MG132in the RPEculture medium.ConclusionsWe concluded that the proteasome inhibitor, MG-132, up-regulates IL-6production in RPE cells through the activation of P38MAPKs. Part II Up-regulation of DR3expression in CD4+T cellspromotes secretion of IL-17in experimental autoimmune uveitisPurpose:This study investigated the role of death receptor3(DR3) in experimentalautoimmune uveitis (EAU).Methods:EAU was induced in B10.RIII mice by subcutaneous injection ofinterphotoreceptor retinoid-binding protein161-180(IRBP161-180) emulsified withcomplete Freund's adjuvant (CFA) and evaluated with clinical and histopathologicobservation. CD4+T cells were separated from lymphocytes with magnetic-assistedcell sorting. At the same time, some of the CD4+T cells were cultured with or withoutrecombinant TL1A (Tumor necrosis factor ligand associated molecular-1A, the DR3ligand) for three days, and the supernatants were collected for the interleukin-17(IL-17) test. DR3mRNA and protein levels in CD4+T cells and the endogenousconcentration of TL1A in mice DLNs were assessed with Real-time PCR or westernblotting. Levels of IL-17in the supernatants were determined with enzyme-linkedimmunosorbent assay.Results:Histopathological and clinical data revealed severe intraocular inflammation in theimmunized mice. The inflammation reached its peak on day14in EAU and hadresolved in the recovery phase (weeks4-5or more after IRBP immunization). CD4+Tcells obtained from EAU (day7or14) had higher levels of DR3mRNA (MessengerRibonucleic Acid) and protein expression compared with the control group treatedwith complete Freund's adjuvant alone and the recovery group. However, the DR3mRNA and protein levels on day21in EAU were similar to those observed in the control and recovery groups. The endogenous levels of TL1A were up-regulated inEAU, and decreased in the recovery phase mice. Adding rTL1A increased theproduction of IL-17by CD4+T cells isolated from mice DLNs. Moreover, theincreased IL-17levels in the culture supernatant of CD4+T cells from EAU weremuch higher than those from the control and recovery phase mice. However, theeffects on promoting IL-17production in TL1A-stimulated CD4+T cells were similarbetween the control and recovery groups.Conclusions:Our data suggest that DR3expression is induced during EAU and may be involvedin the development of this disease, possibly by promoting IL-17secretion.