% deoxycholate, 1 mM PMSF, and Complete Protease Inhibitor Cocktail tablet for 15 min. Detergentinsoluble materials were pelleted at 12,0006g for 15 min at 4uC and supernatant removed. Total protein levels were quantitated using the BCA Protein Assay Kit. Protein samples were separated by 10% SDS-PAGE, transferred to a 0.2 mm polyvinylidene difluoride membrane and immunoblotted with specific antibodies at 4uC O/N. Reactivity was detected with HRP-conjugated goat anti-rabbit S100A12 Blunts Monocyte Cytokine Induction by SAA Assessment of Apoptosis/viability The viability of stimulated THP-1 cells was evaluated using the Annexin V-PE apoptosis detection kit I GW 501516 site following manufacturer’s instructions. Briefly, THP-1 cells were stimulated with SAA, S100A12 or both for,15 h at 37uC in 5% CO2 in air, then washed twice with cold PBS, and resuspended in Annexin V binding buffer. Cell suspensions were transferred to FACS polystyrene tubes, 5 ml Annexin V-PE and 5 ml 7-AAD added, and cells incubated for 15 min at RT in the dark, with gentle shaking. Annexin V binding buffer was added and cells analyzed by flow cytometry immediately after staining. In all experiments, 10,000 events were 6145492 collected from a large gate to exclude debris, but to include all cells. Measurement of TF Procoagulant Activity PBMC were cultured in 250 ml serum-free RPMI 16406 stimulants, in 96-well plates in 5% CO2 in air, as described. After stimulation 8941386 for the times indicated, plates were centrifuged, supernatants discarded, cells resuspended in 250 ml RPMI 1640, then subjected to two cycles of freeze and rapid thawing. Cell-surface TF activity of intact, viable PBMC was measured as described in. Cells were stimulated for the indicated times in Nunc-minisorp tubes, to which monocytes do not adhere, and activity directly measured. PCA was measured using a one-stage plasma recalcification test, using an automatic coagulometer and activity calculated from a standard curve using dilutions of rabbit brain thromboplastin, and expressed as mU TF/106 cells. Statistical Analysis Values in figures are expressed as means 6 SEM. Normal distribution of data was tested and passed the D’Agostino-Pearson normality test. Statistical analyses were performed using a paired t-test or a one-way ANOVA with Bonferroni’s correction for multiple comparisons between groups as indicated. Results S100A12 Suppressed SAA-induced Cytokine Production by PBMC SAA1 and SAA2 are acute-phase SAAs implicated in monocyte activation. S100A12 was also implicated in leukocyte activation and cytokine generation, although our initial results did not reproduce 
these findings. We next considered whether it modified functions of other PAMPs/ DAMPs such as LPS or SAA. The SAA concentration required for cytokine induction was established using PBMC because lymphocytes contribute to optimal TF induction on monocytes by SAA. Since SAA associates with HDL, experiments were routinely carried out in lipid- and serum-free conditions in order to minimize SAA binding to serum proteins. As expected, PBMC cultured in media 6 serum produced higher cytokine levels in serum-free conditions. IL-6 and IL-8 mRNA increased with 1 and 2.5 nM SAA; the suboptimal dose was used in subsequent experiments to allow assessment of positive or negative effects of S100A12. As reported by us, we found no direct induction of proinflammatory cytokines by S100A12. In contrast, S100A12 reduced IL-8 production by SAA-activated PBMC; significant suppression
