WT, Cd11b^–/– (A and B), and Epcr^–/– (C) macrophages, derived by differentiation of BM cells from their corresponding mice, were stimulated by 50 ng/ml LPS in the presence of BSA (as a control) or 0.09 μM hAPC, mAPC, or hGD-APC, with or without NIF (50 nM). Nonstimulated macrophages were used as a control. IL-6 concentration in the conditioned media was determined 20 hours later by ELISA. Data are mean ± SD of 2–3 samples and are representative of 3 independent experiments. *P < 0.01.

(A and B) IL-6 production. WT (A) or Par1^–/– (B) macrophages were treated with 0.09 μM hAPC in the presence of 0.1 mM SCH79797 or 0.25 mM MβCD and stimulated with LPS. IL-6 concentration in the conditioned media was determined by ELISA (n = 3). *P < 0.0001; ^#P = 0.029. (C) Lipid rafts. Murine macrophage RAW264 cells were treated with or without 0.25 mM MβCD and lysed. Cell lysates were separated by sucrose gradient ultracentrifugation, and the presence of CD11b, PAR1, and GM1 (a marker for lipid rafts) within the different fractions was determined by Western blot. (D) Colocalization between CD11b/CD18 and PAR1. BM-derived macrophages were stained with antibodies specific for CD11b (green) and PAR1 (red) and visualized by laser-scanning confocal fluorescence microscopy. Representative images shown were taken with ×100 objective oil lens with a slice thickness of 2.6 μm; an enlarged view of the boxed region in the merged image is shown at bottom right. Scale bars: 20 μm.

(A) BM-derived macrophages from WT, Cd11b^–/–, Epcr^–/–, Par1^–/–, and Sphk1^–/– mice in serum-free media were stimulated with 10 ng/ml LPS in the presence of PBS, 0.09 μM hAPC, or hAPC plus 10 nM NIF for 5 hours at 37°C. S1P production was quantified by ELISA. The amount of S1P in the absence of hAPC treatment was assigned 100%. *P < 0.05 versus PBS. (B) WT, Cd11b^–/–, and Sphk1^–/– macrophages were stimulated with LPS in the presence of PBS, 5 μM FTY720, or 5 μM SEW2871 for 20 hours at 37°C. IL-6 in the conditioned media was quantified by ELISA. Nonstimulated macrophages were used as a control. *P < 0.005 versus PBS. (C) WT or Cd11b^–/– BM-derived macrophages were stimulated with 10 ng/ml LPS in the presence of PBS or 0.09 μM hAPC for 5 hours. Total RNA was prepared, and qRT-PCR was conducted for TRAIL and Wnt5A. All data were normalized to β-actin expression in the same cDNA set. The relative quantity (RQ) values for PBS-treated samples were assigned arbitrarily to 1.0. Relative quantities are mean ± SD of 3 independent experiments. (D) WT and Cd11b^–/– macrophages were stimulated with 10 ng/ml LPS in the presence of PBS, 0.09 μM hAPC, or 5 μM FTY720 for 5 hours, and qRT-PCR was conducted as above using different primer pairs.

(A) Cell adhesion. BM-derived macrophages were allowed to adhere to APC in the presence of 10 nM NIF, 20 μg/ml M1/70, or 40 μg/ml RAP. After washing, adherent cells were counted manually under a microscope. The number of adherent WT macrophages in the absence of inhibitors was assigned 100%. *P < 0.0001 versus unstimulated; ^#P < 0.0005 versus WT. (B) Soluble APC binding. APC was incubated with WT or Cd11b^–/– macrophages in solution. After washing, bound APC was determined by flow cytometry using a goat anti-APC antibody based on MFI. Data are mean ± SD of triplicate samples. (C) Coimmunoprecipitation. CD11b/CD18-expressing or mock-transfected HEK293 cells were treated with APC, washed, and then lysed. The cell lysates were immunoprecipitated using a goat anti-APC antibody, and the pulldown materials were separated on 10% SDS-PAGE and probed with rabbit anti-CD18 antibody ARC22 by Western blot. Equal loading was verified by immunostaining the total cell lysates with anti–β-actin antibody. (D) Colocalization. BM-derived macrophages were treated with APC and then adhered to poly-lysine–coated coverslips. The cells were stained with mouse anti-CD11b/CD18 mAb M1/70 and a goat anti-APC antibody, followed by Alexa Fluor 488–conjugated anti-rat IgG and Alexa Fluor 568–conjugated anti-goat IgG. Specificity was verified using nonimmune rat and goat IgGs (data not shown). Representative images shown were taken with ×100 objective oil lens with a slice thickness of 2.6 μm; an enlarged view of the boxed region in the merged image is shown at bottom right. Scale bars: 20 μm.

(A) Human macrophage THP-1 cells were treated with PBS (open circles) or 20 nM hAPC with (open squares) or without (filled circles) 10 nM NIF at 37°C. (B) THP-1 cells were treated with PBS (open circles), 20 nM hAPC (filled circles), or 1 nM thrombin with (open squares) or without (open triangles) 50 nM NIF at 37°C. At different time points, binding of 0.5 μg/ml ATAP2 to surface PAR1 was determined. The amount of bound ATAP2 at time 0 was assigned 100%. Data are mean ± SD of triplicate experiments. *P < 0.05, hAPC versus hAPC plus NIF.

(A) S1P in plasma. WT and Cd11b^–/– mice (n = 3–4) were injected i.v. with a single dose of 6 μg hAPC or PBS. After 20 minutes, these mice were injected i.p. with 0.8 mg LPS. Blood samples were collected 20 hours later, and concentrations of S1P were determined by ELISA. *P < 0.05 versus PBS. (B) IL-6 in plasma. WT and Cd11b^–/– mice (n = 3) were injected i.p. with 0.8 mg LPS or PBS, followed by i.v. injections with a single dose of 10 μg hAPC, mAPC, hGD-APC, or PBS. Blood samples were collected 20 hours later, and concentrations of IL-6 were determined by ELISA (n = 3–4). *P < 0.05 versus LPS. (C) Endotoxemia-induced mortality. WT (n = 15–49) or Cd11b^–/– (n = 16) mice were injected i.p. with a single LD₉₀ dose of LPS, followed by 2 i.v. injections of 10 μg hAPC (squares), 10 μg hGD-APC (triangles), 10 μg hGD-PC (diamonds),10 μg DFP-hGD-APC (inverted triangles), or PBS (circles) at 20 minutes and 8 hours after LPS challenge. Survival was determined over a period of 14 days and shown in Kaplan-Meier plots. Differences versus PBS were determined by log-rank test for WT (P < 0.0001, hAPC and hGD-APC; P = 0.522, hGD-PC; P = 0.960, DFP-hGD-APC) and Cd11b^–/– (P = 0.585, hAPC; P = 0.497, hGD-APC) mice. (D) Histology. WT and Cd11b^–/– mice were injected i.p. with LPS, followed by i.v. injection of 10 μg hAPC or PBS. Lungs were harvested 20 hours after LPS injection, fixed, and stained with H&E. Images shown are representative of 3 mice. Arrows denote neutrophils; arrowheads denote red blood cells. Scale bars: 20 μm.