E. histolytica host cell killing and phagocytosis are central features of invasive amebiasis. Our study of the role that apoptotic host cell killing plays in amebic phagocytosis had three major conclusions: (i) E. histolytica apoptotic cell killing preceded phagocytosis; (ii) inhibition of the Gal/GalNAc lectin only partially inhibited amebic ingestion of and adherence to apoptotic cells, implicating another receptor in the recognition and clearance of killed cells; and (iii) host cell phosphatidylserine was exposed on the outer membrane of cells killed by E. histolytica, and the surface expression of phosphatidylserine enhanced amebic phagocytosis.
Several experiments indicated the specific recognition and uptake of apoptotic cells by E. histolytica
. Caspase 3 activity was detected in virtually all intact host cells ingested by E. histolytica
. Furthermore, it was possible to partially isolate cell killing from phagocytosis by pretreating amebae with NH4
Cl, which blocks both amebic apoptotic and cytolytic killing of host cells. Under these conditions, E. histolytica
ingested apoptotic cells more efficiently than nonapoptotic cells. Ongoing phagocytosis of healthy Jurkat cells was likely due to an incomplete blockade of amebic cell killing and to the ingestion of cells undergoing spontaneous apoptosis, since NH4
Cl inhibits amebic cytotoxicity by only approximately 40% and approximately 10% of the Jurkat cells used in these experiments were undergoing spontaneous apoptosis in cultures (reference 29
and data not shown). The ingestion of healthy cells was nearly completely eliminated by the more complete inhibition of amebic cytotoxicity by d
-galactose, which blocks the amebic Gal/GalNAc adherence lectin. Importantly, E. histolytica
preferentially ingested apoptotic cells in direct competition with necrotic cells. Taken together, these data indicated that apoptotic killing of host cells by amebae preceded phagocytosis and that the trigger(s) for increased amebic phagocytosis was specific for apoptotic cells and was not simply a general feature of dying cells.
Several experiments implicated at least one amebic receptor in addition to the Gal/GalNAc lectin in the recognition and phagocytosis of apoptotic cells by amebae. The addition of d-galactose at a concentration sufficient to nearly completely block amebic cytotoxicity reduced the phagocytosis of viable cells by approximately 80% but reduced the phagocytosis of already apoptotic cells by only 40%. Furthermore, amebic ingestion of apoptotic cells was not simply a result of increased adherence (e.g., due to more galactose on the dying cells); in fact, approximately 20% fewer amebae adhered to apoptotic cells than to healthy control cells. In the presence of d-galactose, however, significantly more amebae adhered to apoptotic Jurkat cells than to healthy cells. It is important to note that d-galactose did substantially reduce both adherence to and phagocytosis of already apoptotic cells. We concluded that, while the Gal/GalNAc lectin participated in adherence to apoptotic cells during the phagocytic process, at least one additional amebic receptor with a limited role in adherence but the ability to trigger phagocytosis was involved.
The redistribution of phosphatidylserine from the inner to the outer leaflet of the plasma membrane is the most characteristic surface feature of apoptotic cells (12
). Therefore, the transfer of phosphatidylserine from the inner to the outer leaflet of the Jurkat cell plasma membrane was an expected result of apoptotic killing of Jurkat cells by E. histolytica
, and the exposure of phosphatidylserine was confirmed by specific annexin V binding. A significant increase in FITC-dextran staining of Jurkat cells during incubation with amebic trophozoites also occurred and indicated membrane disruption in this subset of cells. The delay in FITC-dextran staining relative to annexin V-FITC staining suggested that membrane disruption might have followed apoptotic killing of these cells (i.e., secondary necrosis). Alternatively, this subset of cells might have been killed by necrosis.
The importance of phosphatidylserine exposure for E. histolytica
engulfment of host cells was suggested by the work of Bailey et al., who previously demonstrated that liposomes containing phosphatidylserine or a synthetic negatively charged phospholipid, dicetyl phosphate, stimulate E. histolytica
actin polymerization (2
). To directly test the ability of host cell phosphatidylserine exposure to stimulate amebic phagocytosis, we quantitated amebic ingestion of viable Jurkat cells following reconstitution of the plasma membrane with phosphatidylserine or several control lipids. Approximately 50% more amebae ingested Jurkat cells expressing phosphatidylserine on the outer leaflet of the plasma membrane than ingested cells coated with phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid. Bailey et al. (2
) suggested that the negative charge on the plasma membrane outer leaflet triggers amebic actin polymerization, so we were surprised to find that phosphatidic acid, another negatively charged phospholipid, had no effect. Unlike the phospholipids used previously, however, phosphatidic acid has no polar head group and no phosphodiester bond. We concluded that the amebic receptor that recognized phosphatidylserine was charge specific and required the presence of a polar head group. In the context of the study of Bailey et al. (2
), however, the receptor is not likely to be a stereospecific phosphatidylserine receptor like that identified in macrophages (13
). Scavenger receptors that mediate the uptake of apoptotic cells in metazoans and that have the ability to bind phosphatidylserine are widely conserved (e.g., Ced-1 in Caenorhabditis elegans
, Croquemort in Drosophila melanogaster
, and CD36 in mammals), suggesting that the amebic phagocytosis receptor may be a scavenger receptor (32
). However, a thorough search of the nearly complete E. histolytica
genome databases (available through The Institute for Genomic Research and the Sanger Centre) identified no amebic homologues (unpublished results).
The nature of the amebic receptor(s) involved in the recognition of apoptotic cells remains an interesting and unresolved issue that we are pursuing. Multiple phagocyte receptors involved in the recognition of apoptotic cells have been described, including a specific phosphatidylserine receptor, lectins, integrins, and scavenger receptors (reviewed in reference 32
). Indeed, the complexity and redundancy of ligands and receptors by which apoptotic cells are cleared in metazoans suggest that multiple amebic receptors and corresponding host cell ligands are likely.
The mechanism by which E. histolytica
cell killing triggers the exposure of host cell phosphatidylserine is also an area of current investigation. Since the caspase 3 inhibitor Ac-DEVD-CHO at a concentration that blocks DNA fragmentation in cells exposed to E. histolytica
did not reduce amebic phagocytosis, ligand exposure during amebic cell killing likely occurs upstream or independently of caspase 3 activation. Phosphatidylserine exposure on dying lymphocytes occurs via both caspase 3-dependent activation of protein kinase C-δ and Ca2+
-dependent activation of a membrane phospholipid scramblase (3
). Furthermore, the inhibition of caspase 3 reduces the quantity of phosphatidylserine exposed on dying lymphocytes but not the number of cells exposing phosphatidylserine (15
). This and prior studies demonstrating a critical role for host cell Ca2+
fluxes early in amebic cell killing suggest a potential mechanism for phosphatidylserine exposure during amebic killing of host cells (28
This study is important because it begins to define a model of sequential E. histolytica
adherence, apoptotic cell killing, and clearance. While a study with cinemicroscopy suggested that amebae kill host cells prior to phagocytosis (as determined by trypan blue uptake), a related study indicated that amebae ingest viable host cells (unstained by trypan blue) when GalNAc lectin-mediated cytotoxicity is blocked (26
). In contrast to these results, our study demonstrated a requirement for host cell apoptosis prior to ingestion by amebae, suggesting that the surface changes characteristic of apoptotic cells (e.g., phosphatidylserine exposure) trigger amebic phagocytosis. The data indicated that E. histolytica
cytotoxicity and phagocytosis are not independent events, as previously concluded (27
). Rather, they are stages in a sequence of events necessary for amebae to cause invasive disease.
Future in vivo studies testing this model may provide insight into why amebic cell killing and phagocytosis are critical to the ability of amebae to cause disease. Although brisk neutrophilic infiltration characterizes early amebic invasion, human autopsy studies have revealed very little reactive inflammation surrounding chronic amebic liver abscesses, and a similar paucity of inflammation has been noted at the base of well-formed colonic ulcers (1
). We hypothesize that the rapid clearance of apoptotically killed cells by amebae (and perhaps also by tissue macrophages) limits the leakage of toxic intracellular contents of killed cells. This effect would be consistent with the paucity of inflammatory infiltration surrounding well-established amebic liver and colonic lesions despite extraordinary tissue destruction and with the ability of E. histolytica
to cause prolonged and/or progressive infection.