Figure 2.

ApoE deficiency or apoE2 expression in macrophages enhances inflammasome priming and activation.A, macrophages isolated from WT and ApoE^−/− (KO) mice (N = 4) were incubated in the absence (−) or presence of 100 ng/ml LPS. Extracts were prepared for Western blot analysis of NLRP3 levels using β-actin as the control. B, macrophages from WT and KO mice (N = 8) were incubated under basal conditions or in the presence of LPS with or without ATP stimulation. IL1-β secreted into the media was measured by ELISA. C, lysates prepared from WT and KO macrophages incubated with LPS with or without ATP were used for Western blot analysis with antibodies against caspase-1 (CASP1) to identify pro-caspase1 and its cleavage products as indicated. Similar experiments were performed with human APOE2, APOE3, and APOE4 gene replacement mice, and results obtained include the following: (D) NLRP3 levels (N = 3); (E) IL-1β secretion under basal conditions (N = 10) or in the presence of LPS with (N = 6) or without (N = 7) ATP; (F) MCP-1 and MIP-1α mRNA levels in LPS-stimulated macrophages; and (G) caspase-1 cleavage analysis in LPS/ATP-stimulated macrophages. Extracts were also prepared from splenocytes isolated from 4 WT and four KO mice (H) or human APOE2, APOE3, and APOE4 gene replacement mice (I) and subjected to Western blot analysis with antibodies against caspase-1 to identify pro-caspase1 and its cleavage product as indicated. All data were evaluated by one-way ANOVA with Student–Newman–Keuls post hoc test for significant differences between groups as indicated. apoE, apolipoprotein E; IL, interleukin; LPS, lipopolysaccharide; NLRP3, NOD-, LRR-, and pyrin domain–containing protein 3.