Monocytes from mutation-positive patients rapidly undergo cell death when treated with LPS. (A) Real-time quantitative reverse transcription (RT)-PCR analysis of CIAS1 mRNA. Purified monocytes were incubated with cLPS (10 ng/mL) or pLPS (10 ng/mL) for 1 hour, and were then subjected to quantitative real-time RT-PCR analysis. Data were normalized to 18S rRNA expression, and represent the means (± SD) of 4 mutation-positive patients or 7 healthy controls. NS: statistically not significant. (B) Intracellular IL-1β staining of PBMCs from CAPS patients. PBMCs from mutation-positive patients (n = 6) or healthy controls (n = 10) were incubated with cLPS (10 ng/mL) for 24 hours. Flow cytometric analysis was performed as described for Figure 1. Representative results of each patient, or the control healthy patients, are shown. Numbers in each quadrant are the percentages of total cells. (C) Monocyte cell number decreases upon LPS stimulation. PBMCs were incubated with or without cLPS (10 ng/mL) for 24 hours, as described in Figure 1. Data represent the number of CD14-positive cells in 10 000 PBMCs, compared with the preincubation state, and represent the means (± SD) of the indicated number of patients. (D) PBMCs from mutation-positive patients were stained with FITC-conjugated anti-CD14, PE-conjugated anti–annexin V, and 7-AAD. Samples were analyzed by flow cytometry, gated to select CD14-positive cells, and then analyzed for cell viability. Data from normal PBMCs that were treated with actinomycin D (AcD, 1 μg/mL) for 12 hours were used as a positive control for apoptotis. Representative data from 5 mutation-positive patients are shown. (E) Giemsa staining of monocytes from mutation-positive patients incubated for 2 hours with or without cLPS. Cells were observed by inverted microscopy using an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with a 40×/0.85 NA objective lens, an Olympus DP70 camera, and DP-controller version 1.1 software (Olympus). Data are representative of 3 mutation-positive patients.