J. Jablonski 1 , E. Jablonska 2 , W. Puzewska 2 , M. Marcinczyk 2 .
Nitric oxide (NO) is a small gaseous molecule with significant bioactivity. Peripheral blood neutrophils (PMNs) are the major participants in a number of pathological conditions with involving NO. NO production by cells requires the presence of a one or more of the nitric oxide synthases (NOS). Inducible type of NOS (iNOS) is usually induced by the presence of certain inflammatory cytokines.
The aim of this study was to examine the NO production and the iNOS expression by PMN and, for comparison, peripheral blood mononuclear cells (PBMC).
Cells were isolated by Gradisol G gradient (1.115g/ml) from 15 healthy donors. The PMN and PBMC were suspended in the culture medium to provide 5x106 cells/ml and cultured for 4h at 37°C in humidified incubator with 5% CO2 (NuaireTM). RhIL-6 (50ng/ml, R&D Systems) and rhIL-15 (50ng/ml, R&D Systems) were tested to stimulate expression iNOS and NO production. Expression iNOS was detected by western blot analysis using mAb capable of detecting iNOS (R&D Systems). NO in the culture of cells was quantified by Griess reagent.
Results obtained indicate that the antibody in human unstimulated and rhIL-6 and rhIL-15-stimulated PMN and PBMC identified band of 130kD from each donors. The rhIL-6 and rhIL-15-stimulated cells expressed a little increased iNOS protein in comparison with unstimulated cells. NO production by rhIL-6 and rhIL-15-stimulated cells was higher than NO production by unstimulated cells. Although observations above do not indicate a significant influence of IL-6 and IL-15 on the iNOS expression and NO production by PMN, the regulation of these proteins by higher concentrations of IL-6 and IL-15 may be of importance for inflammatory and other reactions controlled by these cells.