Figure 4. Mechanism and in vivo role of eicosanoid production.
(a,d) PGE2 immunoassay (30 min) or LDH (2 h) on supernatants from wild-type (B6) resident peritoneal macrophages. Pre-treated 45 min with DMSO, cPLA2 inhibitor (a: pyrrophenone; 0.2 μM) or BAPTA-AM (d: 10μM) then FlaTox + DMSO/inhibitor. (b–c) Wild-type (B6) or Casp1−/− resident peritoneal macrophages treated with FlaTox or FlaTox(AAA) and calcium indicator (Fluo-4; 2.5 mM) fluorescence/background (R/R0) quantified over time. (b) Representative cell, traces. (c) Maximum R/R0 for each cell. (e) Resident peritoneal cells of indicated genotype treated 30 min as indicated. Cell lysates were probed for indicated proteins by Western blot. (f–g) resident peritoneal macrophages (f) or BMDM (g) treated with FlaTox. PGE2 (bars) and cell lysis (line) measured over time as in (a). (h–j) Expression of indicated genes measured by quantitative RT-PCR in BMDM, and resident peritoneal macrophages or thioglycollate-elicited peritoneal cells sorted for CD11b/F4-80hi. (k–n) Mice were injected intraperitoneally with FlaTox (k–l) or anthrax lethal toxin (m–n; 200 μg PA + 100 μg lethal factor) and rectal temperature (k,m) and hematocrit (l,n) were measured after 30 (k–l) or 45 (m–n) minutes. (k–l) B6;129P2-Cox1−/− mice and littermate controls. (m–n) Cox-1−/− mice and littermate controls expressing a lethal toxin sensitive (S) or resistant (R) Nlrp1b allele. Data shown (± s.e.m.) are pooled from multiple experiments (k–n) or representative of at least two (b–c, h–j) or three (a, d–g) independent experiments. * p < 0.04; ** p < 0.006 *** p < 0.0007 (k–n: Mann-Whitney t-test; a,c,d,h–j: Student t-test). # = not detected.