Zileuton (ZL), an inhibitor of 5-lipoxygenase (5-LO) and analog of N-hydroxyurea (HU), decreases the production of inflammatory mediators called leukotrienes in activated polymorphonuclear cells (A-PMN). In asthma, ZL has also been shown to decrease airway inflammation and bronchoconstriction. We have previously demonstrated in isolated rat lung that A-PMN increase sickle red blood cell (SRBC) retention/adherence. In contrast, ZL (2.5 uM) pre-treated A-PMN decreased retention by 43%. These observations led us to ask the questions: 1) does HU decrease SRBC retention/adherence in the rat lung? and 2) does ZL induce fetal hemoglobin (HbF) production? Isolated rat lungs were perfused with 10% SRBC and A-PMN and ventilated with 21% O2-5% CO2. Four groups were studied: 1, SRBC + A-PMN vs SRBC +ZL (2.5(lM) pre-treated A-PMN and 2-4, SRBC + A-PMN vs HU (50 JlM, 100 \lM and 1000 JlM) pre-treated A-PMN. ZL decreased SRBC retention/adherence significantly (p=0.002) and HU at the 1000 |iM concentration decreased SRBC retention/adherence significantly (p=0.008). Experiments were then completed to analyze the ability of ZL to induce HbF. K562 cells were grown in IMDM, 10% fetal bovine serum and ZL at 2.5 u,M to lOOuM concentrations for 72 hours. Total RNA was isolated and y inRNA levels quantitated by RNase protection assay. We observed a 3.3-fold increase in y globin mRNA as a ratio to GAPD at 75|iM ZL compared to a 3.4fold increase for 100 uM HU. K562 cells showed 10% decreased viability at the 75 u,M and 100 |jM concentrations of ZL (comparable to HU). y globin chain biosynthesis was measured at the protein level by alkaline denaturation; a 4-fold increase was observed at the 75 uM ZL concentration. Subsequently we tested the effects of ZL in primary erythroid progenitors from normal controls and sickle cell patients. Peripheral blood mononuclear cells were isolated by hypaque separation and grown in methylcellulose supplemented with interleukin-3 and erythropoietin. A dose dependent 13-fold decrease in BFU-E colony number with ZL at concentrations from 0 to 100 uM and an inverse increase in HbF from an average 3.1% to 19.8% (6.4-fold) were observed for sickle cell samples. Similarly there was a 4.3-fold increase in HbF for normal erythroid progenitors. Summary: 1) ZL decreases SRBC retention mediated by A-PMN in isolated rat lung. HU has a similar effect although at suprapharmacological concentrations. 2) ZL is equally as effective as HU in HbF induction in primary erythroid cultures. Conclusion: Zileuton may prove to be effective in the management of ACS in sickle cell disease.
|Original language||English (US)|
|Issue number||11 PART I|
|Publication status||Published - Dec 1 2000|
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