Effects of TLR ligands and various inhibitors on monocyte cell death. (A) PBMCs from mutation-positive patients or healthy controls were incubated for 24 hours with cLPS (10 ng/mL), MDP (5 μg/mL), Pam3CSK (10 μg/mL), Poly I:C (25 μg/mL), pLPS (10 ng/mL), recombinant flagellin (100 ng/mL), single-stranded RNA (ssRNA, 2 μg/mL), or CpG DNA (1 μM). The number of CD14⁺ cells in 10 000 PBMCs was determined; the ratio of CD14⁺ cells in stimulated PBMCs to those in unstimulated PBMCs is presented. Data represent the means (± SD) of the indicated number of patients or healthy controls (n = 5). (B) Real-time quantitative RT-PCR analysis of CIAS1 mRNA. Purified monocytes were stimulated with TLR ligands or MDP, as described for (A), 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. (C, E) PBMCs from the indicated number of mutation-positive patients or healthy controls (n = 4) were incubated with or without MG-132 (5 μM), YVAD-fmk (50 μM), or CA074-Me (50 μM) for 30 minutes before the addition of cLPS. The cells were then incubated with or without cLPS (10 ng/mL) for 4 hours. The ratio of CD14⁺ and 7-AAD⁻ cells in 10 000 PBMCs relative to untreated cells is shown; data represent the means (± SD) (D, F) Real-time quantitative RT-PCR analysis of CIAS1 mRNA. Purified monocytes were preincubated with the indicated inhibitors for 30 minutes, as described for (C and E), and were then stimulated with cLPS (10 ng/mL) for 1 hour. Thereafter, cells were collected and subjected to quantitative real-time RT-PCR analysis. Data were normalized to 18S rRNA expression. Values represent the means (± SD) of 4 mutation-positive patients or 7 healthy controls. *P less than .05, **P less than .01, NS: not significant, compared with healthy controls by Student t test.