FIGURE 6.

Pre-fibrillar and fibrillar hIAPP aggregates have distinct effects on the synthesis and secretion of IL-1β. A, amyloid fibril content was determined by measurement of thioflavin T fluorescence in freshly dissolved preparations of rIAPP or hIAPP (10 μm) or the mass equivalent of hIAPP that was allowed to pre-aggregate for 24 h at 37 °C (Agg). B, hIAPP (100 μm) was dissolved in water and imaged by TEM after the indicated period of aggregation. Scale bar = 100 nm. C, BMDMs were treated with the indicated stimulus for 3 h (signal 1, to induce proIL-1β synthesis) followed by ATP (5 mm) for 1 h (signal 2, to induce proIL-1β cleavage and mature IL-1β secretion). D, BMDMs were treated with LPS (10 ng/ml) for 3 h (signal 1) followed by the indicated activation stimulus for 16 h (signal 2). E, BMDMs were treated with the indicated priming stimulus (signal 1) for 3 h followed by 1 h or (F) 16 h activation (signal 2) with the indicated form of IAPP (10 μm) or ATP (5 mm). G and H, WT or Nlrp3^−/− BMDMs were treated with the indicated signal 1 for 3 h, followed by the indicated signal 2 for 16 h. IL-1β was detected in cell lysates by Western blot (G) and in supernatants by ELISA (H). Immunoblot data show two different exposures of proIL-1β and are representative of 2 independent experiments; ELISA data show 2 biological replicates per genotype. I, WT or Tlr2^−/− BMDMs were treated as in C to determine the effect of TLR2 on generation of signal 1. J, WT or Tlr2^−/− BMDMs were treated with hIAPP (10 μm) for 0–24 h without priming or (K) following 3 h of pre-treatment with 10 ng/ml LPS to provide an independent source of signal 1. IL-1β in supernatants was measured by ELISA. Data represent mean ± S.D. of 3 replicates per treatment and are representative of 3 independent experiments unless otherwise noted. Dotted line represents the lower limit of detection of the assay. veh: vehicle control. *, p < 0.05; **, p < 0.01; ***, p < 0.001.