Background
Programmed cell death, or apoptosis, is required for the normal development of almost all multicellular organisms and is a physiological mechanism for controlling cell number; as a result, structures that are no longer needed are deleted during development and abnormal cells are eliminated [1,2]. Most of the cells produced during mammalian embryonic development undergo physiological cell death before the end of the perinatal period [3]. Apoptotic cells are removed rapidly and efficiently as intact cells or apoptotic bodies by professional phagocytes or by neighboring cells. This highly regulated process prevents the release of potentially noxious or immunogenic intracellular materials and constitutes the fate of most dying cells throughout the lifespan of an organism [4,5]. Phagocytosis of apoptotic cells is very distinct from other engulfment processes that result, for example, in the clearance of microorganisms, because engulfment of apoptotic cells triggers the secretion of potent anti-inflammatory and immunosuppressive mediators, whereas pathogen recognition causes the release of pro-inflammatory signals [6].
Almost all cell types can recognize, respond to, and ingest apoptotic cells by using specific sets of phagocytic receptors that bind to specific ligands on apoptotic cells. Detailed genetic studies in Drosophila and Caenorhabditis elegans have recently yielded evidence that basic phagocytic mechanisms and pathways for the recognition and engulfment of apoptotic cells are highly conserved throughout phylogeny [7,8]. In vertebrates, a number of receptors have been identified that can mediate phagocytosis of apoptotic cells. These include, for example, scavenger receptors and pattern recognition receptors such as CD36, SR-A and CD14, integrins such as the vitronectin receptor αvβ3, and members of the collectin family and their receptors CD91 and calreticulin [9-13]. The individual roles of these molecules in binding, phagocytosis or transduction of anti-inflammatory signals upon apoptotic cell recognition have not been well defined, however [5,6,14]. The importance of efficient mechanisms for apoptotic cell clearance in vivo is supported by the observation that autoimmune responses can be provoked in mice when key molecules for apoptotic cell recognition and uptake are missing. This has been reported for knockout mice lacking the complement protein C1q [15], for mice with a mutation in the tyrosine kinase receptor gene Mer [16] and, more recently, in mice lacking transglutaminase 2 or milk fat globule epidermal growth factor 8 (MFG-E8) [17,18].
The exposure of the phospholipid phosphatidylserine (PS) in the outer leaflet of the plasma membrane of apoptotic cells has been described as one of the hallmarks of the induction of apoptosis and is considered to be one of the most important signals required for apoptotic cell recognition and removal [19]. A number of cell-surface and bridging molecules can interact with exposed PS on apoptotic cells. These include the serum proteins β2-glycoprotein 1 and protein S [20,21], the growth-arrest-specific gene product GAS-6 [22], complement activation products [23], the milk fat globule protein MFG-E8 [24], and annexin I [25]. In most cases the receptors on phagocytes that recognize these PS-bridging molecules have not been defined, but it has been reported that GAS-6 is a ligand for the tyrosine kinase receptor Mer and that MFG-E8 can bind to the vitronectin receptor αvβ3 [16,24]. Other molecules that bind PS with varying specificity are the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and the scavenger receptors CD36 and CD68 (for review see [5] and references therein).
The best-characterized molecule so far that binds PS in a stereo-specific manner is the phosphatidylserine receptor (Ptdsr) [26]. In vitro, it has been shown that the Ptdsr can mediate the uptake of apoptotic cells and that such Ptdsr-mediated phagocytosis can be inhibited through addition of PS liposomes, the PS-binding molecule annexin V or an anti-Ptdsr antibody [26]. Moreover, the binding of Ptdsr to PS on apoptotic cells has been reported to be important for the release of anti-inflammatory mediators, including transforming growth factor-β1 (TGF-β1), platelet-activating factor (PAF), and prostaglandin E2 [26,27]. These data supported the hypothesis that Ptdsr fulfils a role as a crucial signaling switch after the engagement of macrophages with apoptotic cells and is thereby fundamental for preventing local immune responses to apoptotic cells before their clearance [28].
Very recently, Ptdsr has been found in the cell nucleus. Its nuclear localization is mediated by five independent nuclear localization signals, each of which alone is capable of targeting Ptdsr to the cell nucleus [29]. Moreover, an additional study performed recently in Hydra showed an exclusively nuclear localization for the Ptdsr protein [30]. Most interestingly, the nuclear localization of Ptdsr in Hydra epithelial cells did not change upon phagocytosis of apoptotic cells. These reports challenge the original hypothesis, according to which Ptdsr is an exclusively transmembrane receptor for apoptotic cell recognition and anti-inflammatory signaling.
To examine further the role of Ptdsr in vivo, we performed gene-expression and gene-targeting studies in mice. A perinatally lethal phenotype was observed in Ptdsr-knockout mice, and Ptdsr-deficient embryos displayed multiple defects in tissue and organ differentiation. While this work was in progress, both Li et al. [31] and Kunisaki et al. [32] also reported the generation and phenotypic characterization of Ptdsr-knockout mice. Of note, although some of their results were confirmed in our study, we found a fundamentally different phenotype with regard to clearance of apoptotic cells. Moreover, our study revealed marked and unexpected findings in Ptdsr-deficient mice that are not related to apoptosis.