Differential induction of GM-CSF mRNA accumulation mediated by TNFR1 and TNFR2.

BMSCs were prepared as described in “Materials and methods” and were stimulated with increasing concentrations of rmTNF-α for 2 hours. Northern blots from these experiments are shown on the top; the GM-CSF and cyclophilin (Cyclo) mRNAs are indicated. On the bottom is the PhosphorImager quantitation of these blots after the GM-CSF mRNA levels had been normalized for cyclophilin mRNA levels. WT BMSCs exhibited readily detectable expression of GM-CSF mRNA after stimulation with as little as 0.1 ng/mL rmTNF-α. However, in the absence of TNFR1 (TNFR1KO), there was no induction of GM-CSF mRNA expression, even at 10 ng/mL rmTNF-α. In the absence of TNFR2 (TNFR2KO), rmTNF-α induced the expression of GM-CSF mRNA, but there was a shift to the right of one order of magnitude in the concentration-dependence curve compared with that seen in WT macrophages. The experiment shown here is representative of 2 independent experiments with cells derived from different groups of animals.