SAA induces mature IL-1β synthesis from neutrophils.

<p><b>A</b> Neutrophils were stimulated with the indicated concentrations of SAA for 8-1β production using ELISA. Values represent the mean ± SD of two independent experiments. *<i>p</i><0.001 compared to SAA-untreated neutrophils. <b>B</b> Neutrophils were stimulated with the indicated concentrations of SAA for 8 hr. After stimulation, supernatants were analyzed by western blot analysis for the presence of mature IL-1β. Three experiments were performed using different neutrophils and a representative result is shown.</p

Effects of R406 on NF-κB p65 phosphorylation in SAA-stimulated neutrophils.

<p>Neutrophils pretreated with or without R406 were stimulated with SAA (5 µg/ml) for 20 min. Cellular lysates were analyzed by western blotting using anti-phospho-specific p65 (A) or anti-p65 (B) antibodies. Two experiments were performed using different neutrophils and a representative result is shown.</p

Effects of R406 on the transcription of pro-IL-1β in SAA-stimulated neutrophils.

<p>Neutrophils were pretreated or untreated with the indicated concentrations of R406 for 1(5 µg/ml) for 4 hr. The cells were harvested and analyzed for IL-1β and GAPDH mRNA levels by real-time PCR. Values represent the mean ± SD of three independent experiments. *<i>p</i><0.005 compared to SAA-stimulated neutrophils.</p

SAA induces the transcription of pro-IL-1β in human neutrophils.

<p>Neutrophils were incubated with SAA (5 µg/ml) for the indicated periods. The cells were harvested and analyzed for IL-1β and GAPDH mRNA levels by real-time PCR. Values represent the mean ± SD of three independent experiments.</p

Effects of R406 on the transcription of NLRP3 in SAA-stimulated neutrophils.

<p><b>A</b> Neutrophils were pretreated or untreated with the indicated concentrations of R406 for 1(5 µg/ml) for the indicated periods. The cells were harvested and analyzed for NLRP3 mRNA by RT-PCR. <b>B</b> Neutrophils were pretreated or untreated with the indicated concentrations of R406 for 1 hr. The cells were stimulated with SAA (5 µg/ml) for 4 hr. The cells were harvested and analyzed for NLRP3 mRNA by RT-PCR. Three experiments were performed using different neutrophils and a representative result is shown.</p

SAA-induced IL-1β processing is dependent on caspase-1.

<p><b>A</b> Neutrophils were stimulated with SAA (5 µg/ml) in the presence or absence of Z-YVAD-FMK for 8 h. After stimulation, supernatants were analyzed for IL-1β production using ELISA. Values represent the mean ± SD of two independent experiments. *<i>p</i><0.001 compared to SAA-stimulated neutrophils. <b>B</b> Neutrophils were stimulated with SAA (5 µg/ml) in the presence or absence of Z-YVAD-FMK for 8 h. Supernatants were analyzed by western blot analysis for the presence of mature IL-1β. <b>C</b> Neutrophils were stimulated with SAA (5 µg/ml) in the presence or absence of Z-YVAD-FMK for 8 h. Culture supernatants (SN) and cellular lysates (CL) were analyzed by immunoblot using anti-caspase-1 Ab. Caspase-1 (p20, cleaved subunit; p45, precursor). Two experiments were performed using different neutrophils and a representative result is shown.</p

Effects of R406 on SAA-induced IL-1β processing in neutrophils.

<p><b>A</b> Neutrophils were pretreated or untreated with the indicated concentrations of R406 for 1(5 µg/ml) for 8 hr. After stimulation, supernatants were analyzed by western blot analysis for the presence of mature IL-1β. Three experiments were performed using different neutrophils and a representative result is shown. <b>B</b> Neutrophils were pretreated or untreated with the indicated concentrations of R406 for 1 hr. The cells were stimulated with SAA (5 µg/ml) for 8 hr. After stimulation, culture supernatants (SN) and cellular lysates (CL) were analyzed by immunoblot using anti-caspase-1 Ab. Caspase-1 (p20, cleaved subunit; p45, precursor). Three experiments were performed using different neutrophils and a representative result is shown.</p

Effects of R406 on the protein phosphorylation in SAA-stimulated neutrophils.

<p><b>A</b> Quiescent neutrophils pretreated or untreated with the indicated concentrations of R406 for 1(5 µg/ml) for 20 min. Cellular lysates were subjected to western blotting using phosphotyrosine-specific antibody, 4G10. Three experiments were performed using different neutrophils and a representative result is shown. <b>B</b> Quiescent neutrophils pretreated or untreated with the indicated concentrations of R406 for 1 hr. The cells were stimulated with SAA (5 µg/ml) for 20 min. Syk was immunoprecipitated from each lysates and the immunoprecipitates were subjected to western blotting using phosphotyrosine-specific antibody, 4G10 or anti-Syk antibody. Each lane shows Syk precipitated from 10⁷ cells. Three experiments were performed using different neutrophils and a representative result is shown. <b>C</b> Quiescent neutrophils pretreated or untreated with the indicated concentrations of R406 for 1 hr. The cells were stimulated with SAA (5 µg/ml). Cellular lysates were subjected to western blotting using phospho-specific or pan antibodies against ERK1/2, p38 and JNK1. Three experiments were performed using different neutrophils and a representative result is shown.</p

HLA carrier status in the patients with FMF.

<p>Odds ratio and its 95% confidence intervals (in parenthesis) for the carriers of <i>HLA-B</i> and-<i>DRB1</i> alleles were listed when the comparison with the controls gave p<0.05. The results which remained significant after the Bonferroni’s procedure are highlighted in <b>bold</b> with their p values: 15 <i>HLA-B</i> and 13 <i>DRB1</i> alleles, in total 28 <i>HLA</i> allele carriers were tested because their frequencies in patents and/or controls were 5% or more; p<0.05/28≈0.00179. ns: not significant, (p ≥ 0.05).</p><p>HLA carrier status in the patients with FMF.</p

Interaction between HLA alleles.

<p>^† The effect of carrier status of <i>HLA-B</i> and-<i>DRB1</i> alleles were separately calculated in two subpopulations for different carrier status of the second HLA allele. Odds ratio and its 95% confidence interval (in parenthesis) are given. Mantel-Haenszel weighed mean was given (as shown as adjusted odds ratio in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125938#pone.0125938.t003" target="_blank">Table 3</a>). ns: not significant, (p ≥ 0.05).</p><p>^‡ Test for homogeneity of the effects in two subpopulations. The tests showing differential effects are highlighted in <b>bold</b>.</p><p>Interaction between HLA alleles.</p