Figure 4.

Effect of cyclic AMP-elevating drugs on LPS-induced GM-CSF mRNA expression. Adherent monocytes were pre-treated with salbutamol (5 min; 1 μM), PGE₂ (5 min; 1 μM), 8-Br-cyclic AMP (30 min; 1 mM), rolipram (20 min; 10 μM) or vehicle and exposed to LPS (3 ng ml⁻¹). After 3 h, RNA was extracted and 0.5 μg was reversed transcribed to generate cDNAs for GM-CSF and GAPDH using the primer pairs shown in Table 1. PCR was performed with reverse transcribed cDNA, the products subjected to electrophoresis on 1.5% agarose gels and DNA subsequently visualized after staining with ethidium bromide. PCR products were quantified by Southern blotting and standardized against GAPDH. RT-PCR product sizes for GM-CSF and GAPDH were 500 bp (28 cycles) and 598 bp (24 cycles) respectively. (a) and (b) show the mean data of four independent experiments and a representative gel (prior to Southern hybridization) respectively. 1, Control; 2, LPS+Rolipram; 3, LPS+8-Br-cyclic AMP; 4, LPS; 5, Control; 6, LPS+Salbutamol; 7, LPS+PGE₂; 8, LPS. ^*P<0.05, significant inhibition of LPS-induced GM-CSF mRNA expression.