Milk fat globule-epidermal growth factor-factor VIII-derived peptide MSP68 is a cytoskeletal immunomodulator of neutrophils that inhibits Rac1

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• 作者：Louie Hendricks ; MD^a ; ^b ; Monowar Aziz ; PhD^b ; Weng-Lang Yang ; PhD^a ; ^b ; Jeffrey Nicastro ; MD^a ; Gene F. Coppa ; MD^a ; Marc Symons ; PhD^c ; Ping Wang ; MD^a ; ^b ; ^{pwang@northwell.edu}
• 关键词：Sepsis ; MFG-E8 ; MSP68 ; Lung ; Bone marrow ; Neutrophil ; Small GTPase ; Rac1
• 刊名：Journal of Surgical Research
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：208
• 期：Complete
• 页码：10-19
• 全文大小：649 K
• 卷排序：208

文摘

Prolonged neutrophil infiltration leads to exaggerated inflammation and tissue damage during sepsis. Neutrophil migration requires rearrangement of their cytoskeleton. Milk fat globule-epidermal growth factor-factor VIII–derived short peptide 68 (MSP68) has recently been shown to be beneficial in sepsis-induced tissue injury and mortality. We hypothesize that MSP68 inhibits neutrophil migration by modulating small GTPase Rac1–dependent cytoskeletal rearrangements.MethodsBone marrow–derived neutrophils (BMDNs) or whole lung digest isolated neutrophils were isolated from 8 to 10 wk old C57BL/6 mice by Percoll density gradient centrifugation. The purity of BMDN was verified by flow cytometry with CD11b/Gr-1 staining. Neutrophils were stimulated with N-formylmethionine-leucine-phenylalanine (f-MLP) (10 nM) in the presence or absence of MSP68 at 10 nM or cecal ligation and puncture (CLP) was used to induce sepsis, and MSP68 was administered at 1 mg/kg intravenously. Cytoskeletal organization was assessed by phalloidin staining, followed by analysis using fluorescence microscopy. Activity of the Rac1 GTPase in f-MLP or CLP-activated BMDN in the presence or absence of MSP68 was assessed by GTPase enzyme-linked immunosorbent assay. Mitogen-activated protein (MAP) kinase activity was determined by western blot densitometry.ResultsBMDN treatment with f-MLP increased cytoskeletal remodeling as revealed by the localization of filamentous actin to the periphery of the neutrophil. By contrast, cells pretreated with MSP68 had considerably reduced filamentous actin polymerization. Cytoskeletal spreading is associated with the activation of the small GTPase Rac1. We found BMDN-treated with f-MLP or that were exposed to sepsis by CLP had increased Rac1 signaling, whereas the cells pretreated with MSP68 had significantly reduced Rac1 activation (P < 0.05). MAP kinases related to cell migration including pp38 and pERK were upregulated by treatment with f-MLP. Upregulation of these MAP kinases was also significantly reduced after pretreatment with MSP68 (P < 0.05).ConclusionsMSP68 downregulates actin cytoskeleton-dependent, Rac1-MAP kinase–mediated neutrophil motility. Thus, MSP68 is a novel therapeutic candidate for regulating inflammation and tissue damage caused by excessive neutrophil migration in sepsis.