The efficacy of activated protein C in murine endotoxemia is dependent on integrin CD11b
Chunzhang Cao, Yamei Gao, Yang Li, Toni M. Antalis, Francis J. Castellino, Li Zhang
Chunzhang Cao, Yamei Gao, Yang Li, Toni M. Antalis, Francis J. Castellino, Li Zhang
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Research Article

The efficacy of activated protein C in murine endotoxemia is dependent on integrin CD11b

Abstract

Activated protein C (APC), the only FDA-approved biotherapeutic drug for sepsis, possesses anticoagulant, antiinflammatory, and barrier-protective activities. However, the mechanisms underlying its anti­inflammatory functions are not well defined. Here, we report that the antiinflammatory activity of APC on macrophages is dependent on integrin CD11b/CD18, but not on endothelial protein C receptor (EPCR). We showed that CD11b/CD18 bound APC within specialized membrane microdomains/lipid rafts and facilitated APC cleavage and activation of protease-activated receptor–1 (PAR1), leading to enhanced production of sphingosine-1-phosphate (S1P) and suppression of the proinflammatory response of activated macrophages. Deletion of the γ-carboxyglutamic acid domain of APC, a region critical for its anticoagulant activity and EPCR-dependent barrier protection, had no effect on its antiinflammatory function. Genetic inactivation of CD11b, PAR1, or sphingosine kinase–1, but not EPCR, abolished the ability of APC to suppress the macrophage inflammatory response in vitro. Using an LPS-induced mouse model of lethal endotoxemia, we showed that APC administration reduced the mortality of wild-type mice, but not CD11b-deficient mice. These data establish what we believe to be a novel mechanism underlying the antiinflammatory activity of APC in the setting of endotoxemia and provide clear evidence that the antiinflammatory function of APC is distinct from its barrier-protective function and anticoagulant activities.

Authors

Chunzhang Cao, Yamei Gao, Yang Li, Toni M. Antalis, Francis J. Castellino, Li Zhang

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Figure 5

APC-induced S1P production by activated macrophages is dependent on CD11b/CD18 and SphK1.

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APC-induced S1P production by activated macrophages is dependent on CD11...
(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.