Phagocytosis of microorganisms and “altered self” (e.g.
, apoptotic cells) is important in tissue remodeling and embryogenesis, host-defense and innate immune response, and serves as a nutrient source in unicellular organisms and is required for antigen presentation in mammals. The act of phagocytosis is a sequence of events. It involves the engagement with the object, activation of intracellular signaling pathways, cytoskeleton rearrangement, and internalization. Phagocytized material eventually ends up in phagosomes that fuse with endosomes and lysosomes leading to hydrolytic digestion of their contents. Some microbes have developed mechanisms to avoid their digestion by modifying phagosome maturation, so that this process is also dependent on the cargo it carries (Hornef et al., 2002
). As a phylogenetically old phenomenon, phagocytosis has many redundant pathways, which is one of many reasons why it is so difficult to study. Most investigators interested in phagocytosis focus on the study of cell biology, microscopy and developmental biology and many discoveries were made using receptor expression techniques. The receptors found to be required for the cell surface recognition of pathogens and apoptotic cells involve scavenger receptors (e.g.
CD14 and CD36), complement-like opsonins (e.g.
, C3 and α2
-macroglobulin), multiple EGF-like domains (e.g.
, MEGF10 and CD91), and receptors of the immunoglobulin superfamily.
Using pHrodo-SE as a tag for apoptotic cells to be engulfed by macrophages serves as a novel method for apoptotic cell phagocytosis in vitro
and ex vivo
(). This approach eliminates the possibility of detection of apoptotic cell binding to the surface of the phagocytes and represents an assay equally valid as the use of CAD-/-
thymocytes and TUNEL staining (Hanayama et al., 2002
). Both methodologies use the fact that engulfed material will be biochemically altered after the incorporation into the phagolysosome within the macrophage. In Hanayama's approach (Hanayama et al., 2002
), apoptotic cell DNA is degraded by the phagocyte DNAses while our method uses the pH changes in the endolysosomal microenvironment as an indication for engulfment. The advantage of the present methodology lies in the fact that no knockout animals are needed and this method can be replicated without major difficulties. While this approach has been successfully shown to work in pHrodo-SE-labeled bacteria and latex beads, we show for the first time that it also applicable with apoptotic eukaryotic cells. We have also shown that pHrodo-SE labeled non-apoptotic (live) cells were engulfed by peritoneal macrophages to a significantly lower degree than apoptotic cells. The presence of approximately 5-10% of apoptotic cells in a normal healthy thymus may explain the low-grade phagocytosis in this assay. These results indicate that pHrodo-SE has very minimal effect on phagocytosis. At the same time, we have also addressed the possible limitation of this assay. MFI may have been saturated after a prolonged incubation time (e.g.
, 180min, ). This could be due to the digestion of engulfed apoptotic cells in the phagosome or the decay of the dye.
Flow chart of pHrodo-SE-staining and phagocytosis assay protocol.
We have documented the validity of this method by replicating previous results and demonstrating that MFGE8-deficient macrophages show a delayed and insufficient engulfment of apoptotic cells, which can be recovered by simply adding exogenous recombinant MFGE8 protein. Using this methodology, we also discovered that MFGE8-/-
peritoneal macrophages show a time-delay in apoptotic cell phagocytosis compared to the rmMFGE8-supplemented group, but that these cells eventually catch up and compensate for their deficiency. MFGE8 is one of several molecules that mediate the binding of apoptotic cells to phagocytes. Other molecules include C3b, oxidized low-density lipoprotein (oxLDL), growth-arrest-specific 6 (Gas6), CD14, T-cell immunoglobulin domain and mucin domain 4 (Tim4), etc.
(Savill et al., 2002
; Ravichandran and Lorenz, 2007
). The deficiency of MFGE8 significantly decreases the phagocytic activity of macrophages, but no complete abrogation. Hence, macrophages are still able to phagocytose apoptotic cells via MFGE8-independent pathways that may compensate for the deficient MFGE8 and the suppressed phagocytic activity in our MFGE8 deficient model of peritoneal macrophages we used. The mechanism of these alternative pathways may involve other opsonizing molecules or complement factors and is of great interest, and this new method will serve for further investigations.