Figure 4

Expression ofSPLUNC1in TBE cells grown at the ALI is not induced by pro-inflammatory mediators but requires continued RA driven differentiation. A. TBE cells isolated from three different donors were grown at the ALI as described in the materials and methods section. Cells were treated for increasing lengths of time with 25 ng/ml of IL-1β prior to isolation of RNA. Expression of SPLUNC1 was investigated by the use of RT-PCR with exon spanning primer pairs as described in the materials and methods section. Primers to Elafin and WFDC2 were used as controls. B. The effect of withdrawal of RA from the ALI growth medium was tested in TBE cells from the same three donors. ALI cells were established using standard grow conditions in medium containing 50 nM all trans retinoic acid (RA). After 14 days in culture RA was removed from the medium of one group of cells and culture was continued for an additional 18 days. RNA was harvested at 7 and 14 days during ALI growth and 20, 26 and 32 days following RA removal for 6, 12 and 18 days respectively prior to the isolation of RNA. Expression of SPLUNC1 was investigated by the use of RT-PCR with exon spanning primer pairs as described in the materials and methods section with primers to GAPDH serving as a positive control.