Inhibition of inducible nitric oxide synthase gene expression and nitric oxide synthesis in vascular smooth muscle cells by granulocyte-colony stimulating factor in vitro

Clinical and experimental evidence suggests that granulocyte-colony stimula ting factor (G-CSF) acts as an anti-inflammatory modulator with beneficial effects in severe inflammatory diseases, e.g., sepsis and septic shock. Exc essive production of nitric oxide (NO) is regarded as a potent mediator of the vascular changes leading to systemic hypotension that occurs during sep sis. Therefore, the aim of the present study was to investigate the influen ce of G-CSF on inducible nitric oxide synthase (iNOS) gene expression and N O synthesis in vascular smooth muscle cells (VSMC). Qualitative and quantit ative analyses of iNOS cDNA revealed that G-CSF significantly reduced inter feron-gamma/lipopolysaccharide (IFN-gamma/LPS) dependent iNOS gene expressi on (P < 0.05) following 6, 18, 24, and 48 h incubation periods. In addition , the co-application of G-CSF resulted in a decreased IFN-gamma/LPS mediate d iNOS protein generation as detected by immunoblotting methods after 24 an d 48 h. Measurement of the stable NO metabolites showed a significant reduc tion of nitrite/nitrate concentrations following co-incubation of VSMC with C-CSF + IFN-gamma/LPS (242.57 +/- 10.73 nmol NO2-/NO3-/mg cell protein, n = 8) as compared to IFN-gamma/LPS treatment (306.20 +/- 19.26 nmol NO2-NO3- /mg cell protein, n = 8, P < 0.05) following a 24-h incubation protocol. Th is inhibitory effect of G-CSF was still present after a 48 h incubation per iod (G-CSF + IFN-gamma/LPS: 319.56 +/- 6.26 nmol NO2-/NO3-/mg cell protein; IFN-gamma/LPS: 489.20 +/- 27.15 nmol NO2-/NO(3)(-)mg cell protein (P < 0.0 5), n = 8, respectively). The present findings suggest that inhibition of i NOS gene expression and NO generation in VSMC might be one of the protectiv e anti-inflammatory effects of G-CSF during sepsis. (C) 1999 Elsevier Scien ce B.V. All rights reserved.