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. 2007 Nov-Dec;13(11-12):553-60.
doi: 10.2119/2007-00019.Miksa.

Fractalkine-induced MFG-E8 leads to enhanced apoptotic cell clearance by macrophages

Michael Miksa  1 , Dhruv Amin, Rongqian Wu, Thanjavur S Ravikumar, Ping Wang
Affiliations

Fractalkine-induced MFG-E8 leads to enhanced apoptotic cell clearance by macrophages

Michael Miksa et al. Mol Med. 2007 Nov-Dec.

Abstract

Clearance of apoptotic cells is crucial to maintain cellular function under normal and pathological conditions. We have recently shown that administration of immature dendritic cell-derived exosomes to septic animals promotes phagocytosis of apoptotic cells and improves survival by providing milk fat globule epidermal growth factor-factor VIII (MFG-E8). MFG-E8 acts as an opsonin for apoptotic cells to be engulfed by phagocytosis. In the present study we investigated whether the CX(3)C-chemokine fractalkine (CX(3)CL1) promotes apoptotic cell clearance through the induction of MFG-E8 in peritoneal macrophages. Cultured rat peritoneal macrophages (pMphi) and RAW264.7 macrophages were stimulated with LPS and CX(3)CL1. MFG-E8 expression was assessed by Western blot, cytokine secretion was assessed by ELISA, and phagocytosis of apoptotic thymocytes was determined by microscopy. For in vivo studies, cecal ligation and puncture (CLP) was used to induce sepsis in rats and mice. LPS significantly decreased MFG-E8 levels and phagocytosis of apoptotic cells, whereas CX(3)CL1 induced MFG-E8 expression in both nonstimulated and LPS-stimulated pMphi, without affecting TNF-alpha and IL-6 release. Anti-MFG-E8 blocking antibodies completely abrogated the prophagocytic effect of CX(3)CL1. Twenty hours after the induction of sepsis in rats via CLP, plasma CX(3)CL1 levels as well as MFG-E8 production in peritoneal macrophages decreased by 21% and 56%, respectively. Administration of CX(3)CL1 on the other hand induced MFG-E8 and prevented tissue injury. We conclude that CX(3)CL1 induces MFG-E8 in vitro and in vivo and enhances clearance of apoptotic cells in an MFG-E8-dependent manner. These findings suggest a possible novel treatment for patients in sepsis.

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Figures

Figure 1
Figure 1
Murine recombinant MFG-E8 induces phagocytosis of apoptotic cells by rat peritoneal macrophages. Phagocytosis assay was performed with or without prior opsonization of apoptotic thymocytes with rMFG-E8 (1 μg/mL). Macrophages were incubated with apoptotic cells as targets for 1.5 h, thoroughly washed three times, and stained with TUNEL. (A) Representative fluorescent micrograph showing macrophages in red (PI) and phagocytized apoptotic cells in green (TUNEL). (B) Quantification of phagocytosis index (average control = 1), Means ± SEM, *P < 0.05 vs. control, Student t-test, n = 6. Size bars = 20 μm.
Figure 2
Figure 2
CX3CL1 induces MFG-E8 expression in peritoneal macrophages. (A) 5 × 106 RAW 267.4 macrophages or (B) freshly isolated rat peritoneal macrophages were incubated for 24 h with either LPS (10 ng/mL) or fractalkine (CX3CL1 100 nM) and protein from whole cell lysates was used for MFG-E8 detection by Western blotting. *P < 0.05 vs. control, ANOVA and Student-Newman-Keuls test (SNK), n = 3. (C) CX3CL1 prevents LPS-induced suppression of MFG-E8. Freshly collected rat peritoneal macrophages were stimulated with 10 ng/mL LPS ± treatment with 100 ng/mL fractalkine for 24 h. MFG-E8 protein levels were assessed by Western blotting (representative blots of two separate experiments shown). . *P < 0.05 vs. control, #P < 0.05 vs. LPS alone, ANOVA and Student-Newman-Keuls test (SNK), n = 3.
Figure 3
Figure 3
CX3CL1 promotes phagocytosis of apoptotic cells in peritoneal macrophages. The assay for the phagocytosis of apoptotic cells was performed by incubating freshly collected rat peritoneal macrophages with apoptotic cells for 1.5 h after preincubation with LPS, fractalkine (CX3CL1), or both for 24 h. (A) Immuno-fluorescent micrographs show macrophages in red (PI) with engulfed apoptotic thymocytes in green (TUNEL, size bars = 20 μm). (B) Resident peritoneal macrophages were stimulated with fractalkine (100 nM) for 24 h, washed and blocked with polyclonal anti-MFG-E8 Ab Fab′ (1 μg/mL) for one hour prior to incubation with apoptotic thymocytes for 1.5 h. (C) Phagocytosis index (apoptotic cells/macrophages). Means ± SEM, *P < 0.05 vs. control, # P < 0.05 vs. CX3CL1 only, †P < 0.05 vs. LPS only, ANOVA and Student Newman Keuls’ test, n = 3–5.
Figure 4
Figure 4
CX3CL1 does not induce inflammation in cultured macrophages. RAW264.7 cells (A, B) and freshly isolated peritoneal macrophages (C, D) were stimulated with LPS (10 ng/mL) or fractalkine (CX3CL1, 10 and 100 nM without LPS) for four hours. Panels show concentrations of TNF-α (A, C) and IL-6 (B, D) for both macrophage populations. Means ± SEM, *P < 0.05 vs. control, ANOVA and Student-Newman-Keuls’ test, n = 5.
Figure 5
Figure 5
Sepsis-induced decrease of CX3CL1 and MFG-E8. Rats (A, B) or mice (C, D) were subjected to sham operation or CLP, and blood and peritoneal macrophages were collected at 20 h. (A) Plasma fractalkine (CX3CL1) levels were assessed in blood plasma by ELISA. Means ± SEM, *P < 0.05 vs. Sham, Student t-test, n = 6. (B) MFG-E8 levels in peritoneal macrophages were assessed by Western blotting. A representative blot is shown. Means ± SEM, *P < 0.05 vs. Sham, Student t-test, n = 6. (C) Plasma MFG-E8 levels were assessed using ELISA in septic mice 20 h after CLP with or without concurrent treatment with recombinant murine CX3CL1. (D) Lactate levels were assayed in the same mice using EIA. *P < 0.05 vs Sham, #P < 0.05 vs. Vehicle, ANOVA and Student Newman Keuls’ test, n = 5.
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