Uticaj interleukina-17 na produkciju azot monoksida (NO) od strane L929 ćelijske linije mišijih fibroblasta

Abstract

The ability of interleukin-17 (IL-17) to induce nitric oxide (NO) synthesis in murine L929 fibroblasts was investigated. L929 cells were incubated with IL-17 and/or LPS, interferon-g (IFN-gama), interleukin-1 (IL-1), or dibutyryl-cAMP. NO production was assessed indirectly, by measuring nitrite accumulation in 24 h culture supernatants. L929 fibroblasts did not produce NO constitutively, nor IL-17 alone induced NO production. Of all other stimuli tested, only IFN-gama significantly up regulated nitrite level in L929 cell cultures. However, when IL-17 was applied simultaneously with each of the tested stimuli, NO synthesis was markedly elevated, thus indicating involvement of distinct signaling pathways of NO induction by IL-17 and other tested agents. Production of NO by IL-17+LPS-treated L929 cells was dependent on synthesis and activity of inducible NO synthase (iNOS), as indicated by abrogation of nitrite accumulation with protein synthesis inhibitor cycloheximide or selective inhibitor of iNOS, aminoguanidine. A role for protein tyrosine kinase (PTK) and transcription factor NF-kB in iNOS activation is suggested by reduction of NO synthesis with PTK inhibitor genistein and an inhibitor of NF-kB activation, pyrrolidine dithiocarbamate (PDTC). In contrast, protein kinase C inhibitor staurosporine was ineffective in blocking IL-17+LPS-induced NO production in L929 cells. Taken together, our results for the first time showed an active participation of IL-17 in co-induction of fibroblast NO synthesis with LPS, cytokines (IL-1, IFN-gama), or cAMP analogue, and suggested that IL-17 up-regulated NO synthesis in fibroblasts through mechanisms involving PTK and NF-kB activation.

Ispitan je uticaj interleukina-17 (IL-17) na produkciju azot monoksida (NO) od strane L929 fibroblasta. L929 ćelije su inkubirane sa IL-17 i/ili LPS-om, interferonom-gama (IFN-gama), interleukinom-1 (IL-1), ili dibutiril-cAMP-om. Produkcija NO ispitana je indirektno, merenjem koncentracije nitrita akumuliranih nakon 24 h u supernatantu ćelijskih kultura. Kultivisani L929 fibroblasti nisu produkovali NO konstitutivno, niti je sam IL-17 indukovao NO produkciju. Od ostalih ispitanih agenasa samo je IFN-gama značajno povećao nivo nitrita u kulturama L929 ćelija. Međutim, kada je IL-17 primenjen sa bilo kojim od testiranih agenasa, došlo je do snažnog povećanja NO sinteze, što je ukazalo da su signalni putevi indukcije NO sa IL-17 i ostalim navedenim agensima različiti. Sprečavanje akumulacije nitrita inhibitorom proteinske sinteze cikloheksimidom ili selektivnim inhibitorom iNOS aminoguanidinom u ćelijama stimulisanim sa IL-17+LPS ukazalo je da NO produkcija zavisi od sinteze i aktivnosti enzima inducibilne NO sinteze (iNOS). Inhibitor protein tirozin kinaze (PTK), genistein i inhibitor aktivacije transkripcionog faktora NF-kB, pirolidin ditiokarbamat (PDTC), takođe su redukovali sintezu NO. Nasuprot tome, inhibitor protein kinaze C, staurosporin, nije blokirao NO produkciju L929 ćelija stimulisanih sa IL-17+LPS. U celini, naši rezultati su po prvi put pokazali aktivno učešće IL-17 u koindukciji NO sinteze sa LPS-om, citokinima (IL-1, IFN-gama), ili cAMP analogom u fibroblastima i ukazali da stimulacija NO sinteze sa IL-17 uključuje mehanizme zavisne od PTK i NF-kB.