Phagocytes, including macrophages, recognize phosphatidylserine exposed on apoptotic cells as an "eat me" signal. Milk Fat Globule EGF Factor VIII (MFG-E8) is secreted by one subset of macrophages, whereas Tim4, a type I membrane protein, is expressed by another. These proteins bind tightly to phosphatidylserine on apoptotic cells and enhance their engulfment by macrophages. To study the contribution of these proteins to the engulfment of apoptotic cells, we established a mouse line that was deficient in the genes encoding MFG-E8 and Tim4. The null mutation of Tim4 impaired the ability of resident peritoneal macrophages, but not thioglycollate-elicited macrophages, to engulf apoptotic cells. Mice deficient in either MFG-E8 or Tim4 on the C57BL/6 background developed hardly any autoantibodies, but aged female mice deficient in both MFG-E8 and Tim4 developed autoantibodies in an age-dependent manner. Tumour necrosis factor (TNF) α is known to protect against systemic lupus erythematosus-type autoimmunity, whereas type I IFN accelerates the disease. Indeed, the administration of an anti-TNFα antibody or a reagent that stimulates the IFN-α production [2,6,10,14-tetramethylpentadecane (TMPD; also known as pristane)] enhanced the production of autoantibodies in the MFG-E8- and Tim4-double-deficient mice. These results suggest that the double deficiency of Tim4 and MFG-E8, phosphatidylserine-binding proteins, can trigger autoimmunity and that TNFα and type I IFN regulate reciprocally the development of autoimmune disease.