Fare makrofajlarından nitrik oksit sentezine interferon-gama ve lipopolisakkaritin etkileri

Effects of interferon-gamma and lipopolysaccharide on nitric oxide synthesis from murine macrophages

Özet

Nitrik oksit (NO) makrofajlann sitotoks'ik etkisinin ortaya gikmasmda ml almaktadir. NO'inyaptmmi nitrik ok¬sit sentaz (NOS) enzimi katalize etmektedir. Sitozolde bulunan bu enzimin, enzimi kodlayan genler, hilcre ve-ya organ lokalizasyonlanndakifarkhliklar ve kalsiyum uyartmina verdikleri yanita gore iki izoformu tanim-lanmistir: 1. Uyanlabilir NOS (inducible NOS-iNOS), 2. Yapisal NOS (constitutional NOS-cNOS). Makrofajlar-da iNOS bulunur. Lipopolisakkarit (LPS) ve interferon gamma (IFN-y) iNOS enzimini uyaran maddeler arasm-dadir. Patojen ajanlartn IFN-y tie birlikte makrofajlann NO salgilanmasi uzerine sinerjistik etki yapmalannm yuksek duzeylerde uretildiginde konakgihilcre ve dokulanna karsi da zararli etkileri olabilen NO'in ortamda patojen bir ajan bulunmadikca ilretilmemesi sektinde bir gesii dilzenleyici mekanizma olusturdugu dusunul-mektedir. Ancak, fare kokenli makrofaj tiplerinin IFN-y ve LPS'in tek basina veya beraber uygulanmasi duru-mundaki NO yanitlan, kullamlan hilcre tipleri ve arastmna gruplan arasmda farkhhklar gostermektedir. Bu gahsmada fare makrofaj kokenli RAW264.7 hucresi kullanilarak IFN-y, IPS ve IFN-y+LPS kombinasyonunun NO sentezi uzerindeki etkileri ve bu etkilerin NG-nitrp-L-arjinin-metil ester (L-NAME) ve polimiksin B He inhi-bisyonu arastmldi. Kultiir supernatanlannda NO diizeyi Griess ayiraci He nitrit dilzeyi 6'lgulerek belirlendi. Sa-dece IFN-y ile uyanlan hilcrelerde doza bagimli ve LPS uyanmi He kiyaslanabilir diizeyde NO yanitmin olus-tugu saptandi. IFN-y+LPS kombinasyonu ile uyanlan hilcrelerden, tek basina IFN-y veya LPS ile uyanlanlara gore dahafazla NO salgilanmaktadir. IFN-y veya LPS aracihgi ile NO salgilanmasi L-NAME ile baskilanmakta-dir. LPS'nin biyolojik etkilerini inhibe etmek amaciyla in vitro ortamlarda sikga kullamlan polimiksin B, LPS ile uyanlan hilcrelerdeki NO yanitini inhibe etmenin yanisira, IFN-y ile uyanlan hilcrelerde NO yanitim da anlamli diizeyde (p=0.024) baskilamaktadir.

Abstract

Effects of Interferon-y and Lipopolysaccharide on Nitric Oxide Synthesis from Murine Macrophages Nitric oxide (NO) takes part in emergence ofcytotoxic effect of macrophages. NO production is catalyzed by nit¬ric oxide synthase (NOS). Two isoforms of this enzyme which is found in cytosol have been identified according to the coding genes, differences in cellular or organ localisation and response to calcium stimulation-. 1. Indu¬cible NOS (iNOS), 2. Constitutional NOS (cNOS). Macrophages have iNOS. Lipopolysaccharide (LPS) and inter¬feron gamma (IFN-y) are among iNOS stimulating substances. Synergistic effect of pathogenic agents with IFN-y on NO synthesis in macrophages suggests the presence of a possible regulation system leading to an inhibitory effect when, no pathogenic agent exists on production of NO, that can also be harmful to host cells and tissues when, produced in excessive amounts. However, there are considerable differences in NO responses of murine macrophages to IFN-y, LPS and IFN-y+LPS combination according to the type of the cells and research groups. In this study, using RAW 264.7 murine macrophage cells, effects of LPS, IFN-y and LPS+IFN-y combination on NO synthesis and inhibition of these effects by NG-nitro-L-arjinin-methyl ester (L-NAME) andpolymyxin B we¬re investigated. NO levels in culture supernatants was determined by measuring nitrite levels with Griess re¬agent. Macrophages stimulated only by IFN-y were found to synthesize considerable amount of NO in a dose dependent manner and comparable with LPS stimulation. Macrophages stimulated by LPS+IFN-y combination secreted more NO than the ones stimulated by LPS or IFN-y alone. NO secretion due to IFN-y or LPS was supp¬ressed by L-NAME. Polymyxin B, which is frequently used in in vitro studies in order to inhibit biological effects of LPS, not only inhibited NO response of macrophages stimulated by LPS but also made a considerable supp¬ression of NO response in macrophages stimulated by IFN-y (p= 0.024).