Ptdsr is not essential for the clearance of apoptotic cells
Our studies demonstrate that Ptdsr is not a primary receptor for the uptake of apoptotic cells. Investigation of apoptotic cell clearance in vivo in Ptdsr -/- embryos conclusively showed that removal of apoptotic cells is not compromised by ablation of Ptdsr function. Comparative analysis of ten different tissues and organs in Ptdsr+/+ and Ptdsr -/- animals at several stages of embryonic development and in neonates failed to identify impaired uptake of apoptotic cells at any time during development. Furthermore, phagocytosis assays in vitro demonstrated a completely normal uptake of apoptotic cells by Ptdsr -/- macrophages, with some knockout macrophages showing loads even higher than wild-type of engulfed dead cells. These results are contrary to the expected role of Ptdsr in apoptotic cell clearance and to the reported findings of Li et al. [31] and Kunisaki et al. [32], as well as to a study done with a phosphatidylserine receptor null allele in C. elegans [45]. In previous studies in the mouse, the distribution and amount of apoptotic cells in Ptdsr-knockout and control animals were investigated in only a few tissues and at one [31] or two [32] developmental stages. Li et al. [31] examined lung, midbrain and retina at day E17.5 of gestation and identified apoptotic cells by TUNEL staining. Their findings must be interpreted with caution because remodeling of cellular structures by apoptosis in specific retina layers is known to occur mainly postnatally [42], and apoptosis plays an important physiological role in the maintenance and homeostasis of lung epithelium after birth or in pathological conditions involving pulmonary inflammation and not during lung development [46]. This postnatal role for apoptosis is in accordance with our data, as we rarely observed apoptotic cells in retina or lung tissue throughout embryogenesis in Ptdsr+/+ and Ptdsr -/- mice. Kunisaki et al. [32] analyzed TUNEL-stained sections of liver and thymus at days E13.5 and E16.5 of development in Ptdsr+/- and Ptdsr -/- embryos and found reduced rather than increased numbers of TUNEL-positive cells in Ptdsr-deficient embryos. Using co-localization of TUNEL-positive cells with F4/80-positive macrophages they suggested that Ptdsr -/- embryos exhibited a three-fold increase in the frequency of unphagocytosed TUNEL-positive cells together with a severely reduced number of F4/80-positive cells. These results must be interpreted very carefully, however, as it is technically difficult to unambiguously identify engulfed target cells in individual macrophages in solid tissues by fluorescence microscopy.
In addition, our data suggest that during embryogenesis, macrophage-mediated clearance of apoptotic cells is not the only - or even the primary - mechanism for the removal of apoptotic cells. In many tissues where programmed cell death occurs as a prominent event during embryogenesis, such as remodeling of the genital ridge during gonad morphogenesis and differentiation of the neural tube, we found almost no co-localization of apoptotic cells and macrophages. This indicates that in these cases clearance of apoptotic cells is directly mediated by neighboring 'bystander' cells rather than by macrophages that have been recruited into areas where apoptosis occurs. Obviously these in vivo clearance mechanisms are not compromised by Ptdsr-deficiency in our knockout mutant. This finding is in line with studies in macrophageless Sfpi1-knockout embryos that are deficient for the hematopoietic-lineage-specific transcription factor PU.1. Here, the phagocytosis of apoptotic cells during embryogenesis is taken over by 'stand-in' mesenchymal neighbors [47]. As recognition of phosphatidylserine is thought to be a universal engulfment mechanism for all cells that are able to phagocytose apoptotic cells, it is very striking that apoptotic cell clearance mediated by non-professional bystander cells is also not compromised by Ptdsr-deficiency.
In contrast to Li et al. [31], we did not observe any impairment in the uptake of apoptotic cells by Ptdsr -/- macrophages in vitro. We performed phagocytosis assays in vitro with fetal-liver-derived macrophages, while in their assays, Li and colleagues used thioglycollate-elicited peritoneal macrophages after adoptive transfer of Ptdsr -/- hematopoietic stem cells. The different results obtained in the two studies are puzzling; they might be due to the use of different macrophage or cell populations. We and Kunisaki et al. [32] found that Ptdsr-deficiency is to some extent associated with defects in hematopoiesis. Thus, it seems possible that recruitment and activation/differentiation of macrophages after adoptive transfer and thioglycollate elicitation are affected by Ptdsr-deficiency. We do not think that the different results observed in Ptdsr-knockout mice in a mixed C57BL/6 × 129 background and in a pure C57BL/6J background can be attributed to genetic background effects: comparison of apoptotic cell engulfment efficacies of thioglycollate-elicited macrophages from 129P2/OlaHsd and C57BL/6J mice did not show any differences in apoptotic cell uptake (J.B. and A.L., unpublished observations). Moreover, in contrast to our studies, neither Li et al. [31] nor Kunisaki et al. [32] determined phagocytotic engulfment indexes for Ptdsr-deficient macrophages.
Interestingly, we observed differences between Ptdsr+/+ and Ptdsr -/- macrophages in the secretion of pro- and anti-inflammatory cytokines after stimulation with LPS and apoptotic cells. This provides evidence that cellular activation and effector mechanisms are impaired in Ptdsr-deleted macrophages. It remains to be determined which classical pathways of macrophage activation and function involve Ptdsr. This is especially important in light of recent findings that demonstrated nuclear localization of the Ptdsr protein [29].
Most strikingly, the recently published data regarding the genetic ablation or perturbation of phosphatidylserine receptor function in C. elegans are also contradictory. Wang et al. [45] reported that psr-1, the C. elegans homolog of Ptdsr, is important for cell-corpse engulfment, whereas psr-1 RNAi studies performed by Arur et al. [25] yielded, in this respect, no phenotype. Moreover, Wang and colleagues hypothesized on the basis of their data that psr-1 might act to transduce an engulfment signal upstream of Ced-2 (Crk II), Ced-5 (Dock 180), Ced-10 (Rac 1) and Ced-12 (Elmo) in one of the two cell-corpse engulfment pathways in the worm [45]. But the loss-of-function phenotype of psr-1 mutants and the complementation phenotypes in overexpressing transgenic worms shown by Wang et al. [45] are rather weak as compared to the classical C. elegans engulfment mutants [8].
Many previous functional studies that reported a requirement for Ptdsr for the phagocytosis of apoptotic cells used the monoclonal anti-Ptdsr antibody mAb 217G8E9 [26]. This antibody was used in Ptdsr binding and blocking experiments, as well as in subcellular localization studies, which led to the conclusion that Ptdsr is a transmembrane receptor critical for signal transduction at the engulfment interface. More recently it was used in binding assays to show that the human and worm Ptdsr molecules can recognize phosphatidylserine [45]. In the course of the study presented here, we stained immunohistochemically for Ptdsr with mAb 217G8E9 on wild-type and Ptdsr-deficient macrophages and fibroblasts (see Additional data file 1, Figure S2 and data not shown). To our surprise, we observed similar staining patterns with cells of both genotypes. Furthermore, using a Ptdsr-peptide array we found that mAb 217G8E9 can bind weakly to a Ptdsr peptide, explaining the original isolation of Ptdsr cDNA clones by phage display [26]; but the antibody mainly recognizes additional, as-yet unknown, membrane-associated protein(s) (see Additional data file 1, Figure S2). Experiments that have used this antibody should therefore be interpreted with great caution as they might come to be viewed in a different light.