Expression by RT-PCR of IL-1β, CCL5 and TNF-α in RAW 264.7 cells after sequential treatment with either SP-A followed by TLR ligand (Panel A) or TLR ligand followed by SP-A (Panel B). (A) initial incubation for 2 hours with PBS or SP-A followed by washing and incubation for an additional 5 hours with PBS, LPS, PGN, poly (I:C) or PGN+poly(I:C).(B) initial incubation for 5 hours with PBS, LPS, PGN, poly (I:C) or PGN+poly(I:C), followed by washing and incubation for an additional 2 hours with either PBS or SP-A. P values were calculated by t-test and compare exposures with and without SP-A. n = 3 replicates per condition per experiment. Depicted is a representative figure from three repeat experiments. Error bars = standard deviation. Values for PGN+Poly(I:C) group were set to 100.