FIGURE 7.

β₂GPI binds to fixed viable macrophages in the presence (but not absence) of LPS. B10R or peritoneal (C57BL/6 or TLR4-deficient (TLR4^−/−)) macrophages were fixed and incubated with 20 μg/ml untreated β₂GPI or pmB-B-treated β₂GPI (pmB-β2GPI). Binding of β₂GPI to the macrophage surface was detected by murine anti-β₂GPI mAb (left panels) but was not observed with an isotype-matched control mAb (right panels). Bound murine mAb was detected with FITC-conjugated anti-murine IgG antibody by fluorescence microscopy. Binding of β₂GPI to the macrophage membrane occurred with untreated β₂GPI (upper panel) but not pmB-B-treated β₂GPI (lower panel) in TLR4-sufficient B10R and C57BL/6 macrophages but not in TLR4^−/− macrophages. Micrographs are representative of two independent experiments (magnification ×40) and show an overlay of two acquisitions: FITC staining and Hoechst 33342 staining.