The effect of a new nitro-aspirin on apoptosis of neutrophil granulocytes

Abstract

Apoptosis of neutrophil granulocytes is a critical event in the resolution of inflammation. Neutrophils have a short lifespan which can be modulated by aspirin. In this work we studied the effect of a nitroaspirin (NCX4040) on apoptosis of inflammatory granulocytes. This nitro-aspirin has been synthesized in attempt to reduce the side effects of aspirin in the gastrointestinal tract. Inflammatory granulocytes have been isolated from polyvinyl sponges implanted under the skin of Albino Oxford (AO) rats. Inflammatory cells that were isolated 20 hours later were about 95% neutrophil granulocytes. The cells were cultivated 24h with different concentrations of NCX4040 ranging from 0.01 μM to 10μM. After that period, apoptosis of neutrophils was performed by using morphological criteria, as well as by flow cytometry (after staining the cells with propidium iodide). We found that NCX4040 at concentrations from 0.25 to 10 μM induced the apoptosis of rat inflammatory granulocytes in a dose-dependent manner. Also, in these concentrations NCX4040 decreased production of nitric oxide in the cells culture supernatants. In conclusion, our results suggest that antiinflammatory properties of NO-aspirins are additionally potentiated by their proapoptotic effect on granulocytes, which could be a novel mechanism of their action.

Neutrofilni granulociti su deo imunog sistema, uključeni u odbranu organizma od mikroorganizama. Oni imaju najkraći životni vek među leukocitima, koji se može modulisati citokinima i farmakološkim agensima. Efekat novog derivata aspirina, NCX4040, na apoptozu inflamatornih granulocita do sada nije ispitivan. Zbog toga smo u ovom radu ispitivali efekat nitro-aspirina (NCX4040) na apoptozu inflamatornih neutrofilnih granulocita pacova. Inflamatorni granulociti su izolovani iz polivinilskih sunđera implantiranih potkožno pacovima Albino Oxford (AO) soja. Inflamatorne ćelije koje su izolovane 20 sati kasnije, najvećim delom (više od 95 %) su bili neutrofilni granulociti. Ove ćelije su kultivisane 24 sata sa NCX4040 u koncentracijama od 0.01 μM do 10 μM. Supernatanti kultura neutrofila su sakupljani, i korišćeni za merenje koncentracije NO. Ćelije su bojene propidijum jodidom i apoptoza je analizirana na protočnom citofluorimetru, kao i pomoću morfoloških kriterijuma. Ustanovili smo da u koncentracijama od 0.25 μM do 10 μM ovaj nitro-aspirin indukuje, na dozno zavisan način, apoptozu neutrofilnih granulocita. Apoptoza indukovana nitro-aspirinom je bila u pozitivnoj korelaciji sa smanjenom produkcijom NO.