Uticaj metilprednizola na ekspresiju i produkciju interferona-Θ i interleukina-17 u eksperimentalnom autoimunskom encefalomijelitisu

Abstract

Uvod: Th17 ćelije predstavljaju populaciju ćelija koje imaju ulogu u imunopatogenezi autoimunskih oboljenja centralnog nervnog sistema kao što su multipla skleroza i eksperimentalni autoimunski encefalomijelitis (EAE). Efekat glukokortikoida, lekova koji se primenjuju kao terapija inflamatornih i autoimunskih oboljenja, na IL-17 još uvek nije ispitan. Stoga je u ovoj studiji ispitivano dejstvo sintetskog glukokortikoida (GK), Metilprednizolona (MP) na ekspresiju i produkciju IL-17. Kako sam mehanizam i mesto dejstva GK nije precizno definisan, takođe je ispitan in vivo efekat MP na ekspresiju i produkciju IL-17 od strane mononuklearnih ćelija kičmene moždine (MNĆKM) tretiranih DA pacova tokom eksperimentalnog autoimunskog encefalomijelitisa (EAE). Materijal i metode: EAE je indukovan kod Dark Agouti (DA) pacova sa homogenatom kičmene moždine uz kompletni Frojndov adjuvans (CFA). Počevši od prvog dana bolesti, DA pacovi su tretirani sa MP i/ili antagonistom glukokortikoidnog receptora- RU486. Produkcija citokina od strane ćelija koje infiltriraju CNS kao i ekspresija gena je merena ELISA metodom i kvantitativnim PCR-om. Rezultati: MP inhibira produkciju IL-17 od strane mitogenom stimulisanih ćelija limfnog čvora (ĆLČ) DA pacova kao i antigen specifičnu produkciju od strane ćelija drenirajućeg limfnog čvora. Inhibicija produkcije IL-17 je dozno zavisna i nije uslovljena inhibicijom proliferacije tokom 48h inkubacije. Takođe, MP inhibira produkciju IL-17 od strane prećišćenih T limfocita, ali ne tako izraženo kao u slučaju ĆLČ DA pacova. Sa druge strane pokazano je da MP ublažava kliničko ispoljavanje EAE-a što je praćeno i inhibicijom ekspresije i produkcije IL-17 od strane mononuklearnih ćelija kičmene moždine tretiranih DA pacova. Zapažen efekat MP primenjenog in vivo nije posledica smanjenja procentualne zastupljenosti CD4+ T limfocita kao ni njihove apoptoze. Na kraju, antagonist glukokortikoidnog receptora Mifepriston- RU486 poništava inibitorno dejstvo MP na ekspresiju i produkciju IL-17 u in vitro i u in vivo uslovima, što ukazuje da su zapaženi efekti MP posredovani putem glukokortikoidnog receptora...

Background: Interleukin-17 (IL-17)-producing cells are increasingly considered to be the major pathogenic population in various autoimmune disorders. The effects of glucocorticoids, widely used as therapeutics for inflammatory and autoimmune disorders, on IL-17 generation have not been thoroughly investigated so far. Therefore, we studied the effects of a synthetic glucocorticoid methylprednisolone (MP) on IL-17 expression and production in rat lymphocytes. Since the mechanisms and the site of glucocorticoids' actions are still not completely defined we also investigated the in vivo effect of the synthetic glucocorticoid MP on the expression and production on IL-17 by cells infiltrating CNS tissue during experimental autoimmune encephalomyelitis (EAE). Methods: EAEwas induced in Dark Agouti (DA) rats by immunization with rat spinal cord homogenate (SCH) mixed with adjuvant. Commencing on the day when the first EAE signs appeared, DA rats were injected daily for 3 days with MP and/or RU486, an antagonist of glucocorticoid receptor. Cytokine production and gene expression in CNS infiltrating cells and lymph node cells were measured using ELISA and real time PCR, respectively. Results: Production of IL-17 in mitogen-stimulated lymph node cells (LNC) from nontreated rats, as well as in myelin basic protein (MBP)-stimulated draining LNC from rats immunized with SCH + adjuvant was significantly reduced by MP. The reduction was dose-dependent, sustained through the follow-up period of 48 hours, and was not achieved through anti-proliferative effect. Additionally, MP inhibited IL-17 production in purified T cells as well, but to less extent than in LNC. On the other hand treatment of rats with MP ameliorated EAE, and the animals recovered without relapses. Further, MP inhibited IL-17 expression and production in cells isolated from the CNS of DA rats with EAE after the last injection of MP. The observed effect of MP in vivo treatment was not mediated through depletion of CD4+ T cells among CNS infiltrating cells, or through induction of their apoptosis within the CNS. Finally, the glucocorticoid receptor-antagonist RU486 prevented the inhibitory effect of MP on IL-17 production both in vitro and in vivo, thus indicating that the observed effects of MP were mediated through glucocorticoid receptor-dependent mechanisms...