Figure 1.

Cystine crystals induce IL-1β secretion in LPS-primed human PBMCs and are internalized by monocytes in vitro. (A) Primary human PBMCs were primed with LPS (100 ng/ml) for 2 hours and then incubated with increasing concentration of cystine crystals or monosodium urate crystals (MSU) for 6 hours. Supernatants were collected and IL-1β was measured by ELISA. Data are representative of three independent experiments performed in duplicate and are expressed as means±SD (top). Supernatants (SN) were also analyzed by Western blotting using antibodies detecting the mature form of IL-1β and the activated caspase-1 form (bottom). (B) Monocytes isolated by adherence from freshly isolated PBMCs were prestimulated with LPS (100 ng/ml) for 2 hours and then incubated with cystine crystals for 6 hours. At the end of incubation, cells were stained with anti–wheat germ agglutinin (WGA) lectin (green) to label plasma cell membranes and Hoechst stain (blue) to label nuclei. Cells were imaged by confocal fluorescent microscopy. (b–e) Single section performed on the central focal plane, showing wheat germ agglutinin staining of cell membranes (b), cell phase contrast (c), wheat germ agglutinin and Hoechst staining (d), and merge image (e) visualizing cystine crystals enveloped by cell membranes. Cystine crystals are indicated by arrows. Scale: 50 μm (a) and 10 μm (b–e).