Supplementary Figure 1: Leukocyte expression of IL-18R, BAFF and Fas, plus evaluation of Pad4^–/– mice.

(a) Flow cytometry detection of IL-18R on mouse naïve splenic neutrophils and follicular B cells. (b) Quantitative RT-PCR analysis of mRNA encoding BAFF (Tnfsf13b) in sorted splenic macrophages freshly isolated from vehicle or IL-18 injected mice at day 12. Results are normalized to mRNA encoding Hprt1 to determine mean fold change, and are presented as relative expression (RE) compared with splenic macrophages after vehicle treatment. (c) Flow cytometry of neutrophils cultured 16 h in vitro with medium (left panels) or 20 ng/ml IL-18 (right panels). Neutrophils were labeled with isotype control (top panels) or anti-BAFF antibody (clone 121808; bottom panels). Boxes indicate BAFF⁺ neutrophils. (d) Flow cytometry of frequency of CD11b⁺ Ly6G^—macrophages (MØ) expressing surface BAFF following 16 h in vitro culture in the presence of 20 ng/ml IL-18 or medium. (e) Flow cytometry determination of frequencies of splenic B220⁺ B cells expressing Fas following co-culture with or without neutrophils and 20 ng/ml IL-18 or medium for 6 h, as noted. Anti-BAFF antibody or BAFF–R–Ig was added to the cultures to block BAFF. (f) Confocal immunofluorescence of neutrophil elastase in the spleens of wild-type (C57BL/6) mice treated with vehicle (left) or IL-18 (right) at day 12. (n = 3 per group). Scale bar, 100 μm. (g) ELISA measure of serum titers of total IgE and (h) anti-DNA IgM, IgG, and IgE antibodies (day 12) in homozygous Pad4^–/– mice and heterozygous littermate controls on day 12 after injections with IL-18 or vehicle. *P < 0.05 [two-tailed unpaired Student’s t-test (b; n = 4 per group, 3 technical replicates), (d, n= 6 per group, 3 technical replicates), 2way ANOVA with Sidak correction (e, n= 5 per group, two technical replicates) or (g, h; n = 5 per group). Data are representative of one (f), two (e), three (c) experiments or pooled from two (d) or five experiments (g, h). Box height indicates mean + s.e.m.