Abstract
Milk fat globule epidermal growth factor 8 (MFG-E8) is a protein that binds to apoptotic cells by recognizing phosphatidylserine and enhances the engulfment of apoptotic cells by macrophages. Many apoptotic cells are left unengulfed in the germinal centers of the spleen in the MFG-E8-deficient (MFG-E8−/−) mice, and these mice develop an autoimmune disease resembling human systemic lupus erythematosus. We found that the MFG-E8 deficiency was accompanied by the increased production of immunoglobulins. Further Western blot and ELISA analyses validated the increase in the IgM levels in the MFG-E8−/− mice. It was also revealed that the sera from the MFG-E8−/− mice cross-reacted with oxidation-specific epitopes generated upon incubation of serum albumin with the peroxidized lipids. Among the modified proteins with several unsaturated aldehydes of chain lengths varying from three to nine carbons, the MFG-E8−/− mice sera exclusively cross-reacted with the protein-bound 4-oxo-2-nonenal (ONE), a highly reactive aldehyde originating from the peroxidation of ω6 polyunsaturated fatty acids. In addition, the IgM monoclonal antibodies (mAbs) that selectively cross-reacted with the ONE-modified proteins were generated from the MFG-E8−/− mice. A subset of the ONE-specific IgM mAbs significantly recognized the late apoptotic and necrotic cells and enhanced the phagocytosis by macrophages. These data demonstrate that the impairment of the phagocytic clearance of apoptotic cells through MFG-E8 can lead to the generation of natural antibodies, which may play a critical role in removing multiple damage-associated molecules, including oxidation-specific epitopes and late apoptotic/necrotic cells.
Highlights
Milk fat globule epidermal growth factor factor 8 (MFG-E8), originally found associated with milk fat globules in mammary glands, is a secreted protein present on a subset of phagocytes that actively engulf apoptotic cells [1]
When the MFG-E8+/+ and MFG-E82/2 mice sera were analyzed by an immunoblot analysis with the anti-whole IgG Ab, a significant increase in the proteins cross-reacted with the Ab was observed in the MFG-E82/2 mice (Figure 2A)
Among the immunoglobulins (IgA, IgD, IgG, and IgM) tested, both IgA and IgD were nearly undetectable in both groups, whereas IgG was detectable in the MFG-E8+/+ mice and its level increased in the MFG-E82/2 mice
Summary
Milk fat globule epidermal growth factor factor 8 (MFG-E8), originally found associated with milk fat globules in mammary glands, is a secreted protein present on a subset of phagocytes that actively engulf apoptotic cells [1]. It is expressed by macrophages and immature dendritic cells, including tingiblebody macrophages and follicular dendritic cells at the germinal centers in the spleen and lymph nodes, thioglycollate-elicited peritoneal macrophages, granulocyte-macrophage colony stimulating factor-induced bone marrow-derived immature dendritic cells, and Langerhans cells in the skin [2,3,4]. MFG-E8 associates with the avb or avb integrin on phagocytes via its RGD motif [6], binds tightly to phosphatidylserine through its C1 and C2 domains, and stimulates the engulfment of apoptotic cells (Figure 1) [1]
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