Analysis of iNOS expression in PB-NK and S-NK cells. (A) Expression of iNOS mRNA in PB-NK and S-NK cells after treatment with IL-2. The mRNA of stimulated and unstimulated cells was isolated and after RT-PCR was performed with specific primers for iNOS and β-actin. Lane 1, untreated rat peritoneal macrophages; lane 2, rat peritoneal macrophages stimulated with IFN-γ plus LPS for 18 hours; lane 3, control S-NK cells; lane 4, S-NK treated with IL-2 for 96 hours; lane 5, untreated PB-NK; lane 6, PB-NK treated with IL-2 for 18 hours. Size marker: DNA molecular weight marker VI (Boehringer) (M). Data show one representative experiment of three. (B) Western blot analysis with specific anti-iNOS MoAb in 200 μg of total cell lysates obtained from PB-NK cells or S-NK. As internal control for iNOS, 50 μg of rat peritoneal macrophage lysates were loaded into the same gels. Lane 1, untreated PB-NK; lane 2, PB-NK treated with IL-2 for 18 hours, lane 3, control S-NK cells; lane 4, S-NK treated with IL-2 for 96 hours; lane 5, untreated rat peritoneal macrophages; lane 6, rat peritoneal macrophages stimulated with IFN-γ plus LPS for 18 hours. (C) iNOS expression on CD161 positive PB- and S-NK cells after treatment with IL-2 as above described. Two-color cytofluorographic analysis was performed using mouse anti-rat CD161–PE MoAb and mouse specific anti-mouse macNOS MoAb and FITC-conjugated GM. As control antibodies, anti-human CD56-PE MoAb and GM-FITC MoAb were used.