Rat macrophages were pretreated by each hormone (1000 ng/mL of CORT, E, and NE) for 24 h, then restimulated with Pam3CSK4 or lipopolysaccharide (LPS) for further 24 h, and supernatant tumor necrosis factor-alpha (TNF-伪) was measured. Additionally, macrophages were incubated with different concentrations of hormones (0-10,000 ng/mL) for 48聽h or with 1000 ng/mL of hormones for 0-48 h, the expressions of TLR2 and TLR4 and intracellular molecules (MyD88, IRAK1, and TRAF6) in macrophages were analyzed by real-time quantitative polymerase chain reaction (PCR) and RT-PCR, respectively.
Pam3CSK4-stimulated TNF-伪 production was significantly reduced from macrophages pretreated with CORT, and both Pam3CSK4- and LPS-stimulated TNF-伪 were suppressed with E. Moreover, CORT down-regulated only TLR2 expression in both time- and dose-dependent manner, but both TLR2 and TLR4 mRNA expressions were down-regulated in time- and dose-dependent manner after exposure to E. However, the transcript expression of MyD88, IRAK1, and TRAF6 remained unchanged after exposure to each hormone.
These results suggested that the down-regulation of TLR2 and TLR4 expressions by CORT and E is involved in the hyporesponsiveness of macrophages.