Murine macrophage MIF production, by LPS-activated cells, is decreased with concurrent estrogen treatment (a). Results represent means ± SEM (n = 10 for each group) *P < 0.05, **P < 0.01, all compared with control media plus LPS-activated macrophages (C+). LPS activation significantly increased MIF production as compared with control media alone (P < 0.05). (b) Immunostaining illustrates that monocytes (CD14+ cells) express both ER-α and ER-β isoforms. The far right panel of b represents a merged image of all three panels (ER-α, ER-β, and CD14). Scale bar represents 10 μm. (c) LPS treatment increases MIF levels from wild-type murine cells and is inhibited by estrogen. In the absence of ER-α (ER-α null), estrogen fails to downregulate LPS-induced MIF production. LPS significantly increased MIF in both wild-type and ER-α null macrophages (P < 0.05). Seeding density of cells is three times higher than in Figure 5a. Results represent means ± SEM (n = 5 for each group, *P < 0.05 as compared with LPS-activated cells). (d) MCF7 ER-expressing cells transfected with a MIF promoter–luciferase reporter construct showed increased activity after LPS activation (a). Estrogen (10–8 M) inhibited this activity. Transfection with pGL3 basic plasmid acted as a negative control (pGL3–ve), and pGL3 control plasmid as a positive control (pGL3+ve). Results represent means ± SEM (n = 3–5 for each group, *P < 0.05 as compared with LPS-activated cells). (e) MCF7 ER-expressing cells transfected with a MIF promoter–luciferase reporter construct showed inhibition of LPS-activated MIF production by estrogen and estrogen agonists (PPT, HPTE) and reversal of estrogenic effects by ER antagonist (ICI). Results represent means ± SEM for three experiments (*P < 0.05 as compared with LPS-activated cells). C, control media alone; +, + LPS treatment for 6 hours; E8, 10–8 M estrogen; E9, 10–9 M estrogen; E10, 10–10 M estrogen; E11, 10–11 M estrogen; RLU, relative light units.