Figure 6.

Silencing iNOS transcription decreases attenuation of GM-CSF message and protein release elicited by (R)-albuterol. (a) NHBE cells were transfected for 24 h with iNOS-silencing RNA, as described in the text. After the 24-h transfection period, cells were stimulated with 10 ng/ml each of IL-1β and IFN-γ for 18 h to augment GM-CSF message levels. iNOS silencing had little effect on IL-1β + IFN-γ augmentation of GM-CSF message (lanes 1–4), but it did negate (R)- and racemic albuterol–mediated suppression of GM-CSF message (lanes 5–8). (S)-albuterol, with or without silencing, did not affect GM-CSF message expression (lanes 9 and 10). These results suggest that iNOS is necessary for (R)- and racemic albuterol–mediated GM-CSF message suppression in NHBE cells exposed to cytokines. Dashed line represents constitutive GM-CSF expression. Data are presented as mean ± SEM (n = 3–4). ^* Significantly greater than constitutive GM-CSF expression (P < 0.05, ^** = P < 0.001); ^† significantly lower than GM-CSF expression after treatment with iNOS silencing (P < 0.01); ^§ significantly lower than cytokine-stimulated GM-CSF expression (P < 0.05). N.S., no significant difference (P > 0.05). (b) NHBE cells were transfected and treated as described previously here, and GM-CSF protein in culture medium was measured by ELISA. The addition of iNOS small interfering RNA (siRNA) did not significantly affect cytokine-mediated GM-CSF protein release. However, (R)-albuterol (10⁻⁵ M) significantly suppressed cytokine-mediated GM-CSF protein release, and iNOS silencing partially suppressed this effect. Dashed line represents constitutive GM-CSF protein release. Data are presented as mean ± SEM (n = 12). ^** Significantly greater than constitutive GM-CSF protein release (P < 0.001); ^†† significantly lower than cytokine-induced GM-CSF protein release (P < 0.001); ^§ significantly greater GM-CSF protein release compared with cytokine + fugene 6 + (R)-albuterol–treated cell cultures (P < 0.05).