Apoptosis occurs throughout the lifetime of an organism, from embryogenesis to death. Recent evidence lends further support to the concept that efficient removal of apoptotic cells and cell bodies prevents exposure of surrounding tissue to potentially cytotoxic, immunogenic, or inflammatory cellular contents (Albert et al., 1998
; Sauter et al., 2000
). The removal process is completed by a wide variety of cell types and appears to involve multiple receptors and apoptotic cell ligands, yet the phagocytic mechanism has not been clearly defined. The data presented here suggest that uptake of apoptotic cells involves two separate steps: an initial tethering event followed by PS-stimulated macropinocytosis.
Many putative phagocytic receptors appear to mediate tethering of the apoptotic cell to the phagocyte. In contrast, PS by itself appears to promote poor binding of particles to phagocytes, but plays a critical role in stimulating the actual uptake process through the PSR. The ability of PS to drive internalization of any tethered particle may, in part, explain the recent reports that apoptotic and necrotic cells appear to be recognized and engulfed in a similar manner (Chung et al., 2000
; Cocco and Ucker, 2001
). In this proposed scenario, binding and ingestion are separable events. However, the requirement for both a tethering receptor and an engulfment receptor is indispensable. It is plausible that the ability of macrophages, or professional phagocytes, to rapidly and efficiently clear apoptotic cells compared with their nonprofessional counterparts may be due to the wide repertoire of adhesion receptors, not to differences in engulfment mechanisms (Parnaik et al., 2000
). In addition, we have recently suggested that the collectin family of innate immune system molecules can mediate uptake of apoptotic cells (Ogden et al., 2001
), perhaps because they can enhance adhesion to phagocyte surfaces. However, surface calreticulin, a receptor for the collagenous domains common to the collectin family, also appears to be capable of stimulating macropinocytosis, perhaps through CD91 (α2 macroglobulin receptor/LRP), and may provide another example of apoptotic cell removal by this mechanism (Ogden et al., 2001
The requirement for Rac1 during anti-PSR antibody-mediated membrane ruffling suggests that the PSR utilizes signaling pathways for apoptotic cell engulfment similar to those defined in the Caenorhabditis elegans
system (Gumienny et al., 1999
; Liu and Hengartner, 1999
; Chung et al., 2000
; Zhou et al., 2001
). In addition, the PSR appears to require the activity of Cdc42 for the induction of membrane ruffling. How the PSR transmits signals to the low molecular weight GTPases is currently unclear. Although our results suggest that receptors such as SRA, αv
integrins, and CD36 do not transmit engulfment signals, these studies do not rule out the possible formation of a complex, where tethering receptors and the PSR act in concert to transmit engulfment signals. The integrin, αv
has been shown to recruit Rac1 and Dock180 via the adaptor protein CrkII in HEK293 cells in the presence of apoptotic lymphocytes (Albert et al., 2000
). It is interesting to speculate that the integrins implicated in apoptotic cell engulfment may be activated by the binding of target ligands on the apoptotic cell, resulting in the recruitment of Rac1 to the engulfment site and the induction or amplification of actin cytoskeleton rearrangement.
Surprisingly, PS alone on the surface of erythrocytes resulted in limited adhesion to the phagocyte surface. The lack of particle binding by PS–PSR interactions could reasonably be explained by either low surface expression of the PSR on the phagocytes, i.e., low avidity, or by low affinity between PS and the PSR. HMDM from many donors have been shown to express the PSR at low levels unless stimulated with TGF-β and β-glucan (Fadok et al., 2000
). However, preliminary studies using HMDM, stimulated to upregulate PSR expression, have not resulted in increased erythrocyte binding, suggesting that low avidity is not responsible for the lack of PS-coated erythrocyte binding. Affinity studies are currently being conducted to determine the strength and specificity of the PS–PSR interactions. It is imagined that relatively low affinity PSR interactions with PS are markedly enhanced by localized expression of PS on the apoptotic cell and, in particular, by the binding of the apoptotic cell by recognition receptors.
As macropinocytosis represents a nearly universal mechanism for ingestion by eukaryotic cells, it follows that PS may be capable of initiating this mechanism in a wide variety of cell types. It is tempting to speculate that the presence of PS is a powerful and ubiquitous engulfment signal, capable of stimulating the ingestion of apoptotic cells by many different types of phagocytes, regardless of the initial tethering ligand. These data suggest that PS stimulation of macropinocytosis and apoptotic cell engulfment is mediated in majority by the PSR. The PSR appears to be expressed by nearly all tissue types, and PSR homologues have been identified not only in man and mouse but also in Drosophila melanogaster
and C. elegans
(Fadok et al., 2000
). Although playing a minor role in the binding of apoptotic cells, the PSR is critical for their ingestion, as indicated by antisense transfection data. In addition to conducting a signal that contributes to apoptotic cell uptake, the PSR appears to be responsible for inducing the release of a key antiinflammatory, antiimmunogenic molecule, TGF-β. The structure, localization, and characterization of domains on the PSR that mediate signaling for phagocytosis and cytokine production are currently under investigation.