Differential regulation of phagosome maturation in macrophages and dendritic cells mediated by Rho GTPases and ezrin-radixin-moesin (ERM) proteins

Abstract

Deletion of apoptotic cells from tissues involves their phagocytosis by macrophages, dendritic cells, and tissue cells. Although much attention has been focused on the participating ligands, receptors, and mechanisms of uptake, little is known of the disposition of the ingested cell within the phagosome. Here we show that uptake of apoptotic cells by macrophages or fibroblasts results in rapid phagosome maturation, whereas macrophage phagosomes containing Ig-opsonized target cells mature at a slower rate. The early maturation was shown to depend on activation of Rho acting through Rho kinase on ezrin-radixin-moesin proteins. Blockade of Rho signaling or inhibition of moesin both delayed maturation rates to those seen with opsonized targets. By contrast, phagosome maturation in dendritic cells was slower, similar between apoptotic and opsonized target cells, and unaffected by Rho inhibition. These observations have direct implications for the clearance of dying cells and the roles played by different phagocytes in antigen digestion and presentation.

Fig. 1.

Acidification of phagosomes containing apopotic and opsonized cells. (A) Image of PKH-labeled apoptotic neutrophils colocalizing with LTR at 90 min in J774. Noningested attached cells (arrow) do not colocalize. (B and C) Colocalization of ingested cells with LTR or lysosensor. (D) Rate of uptake expressed as phagocytic index. (E) Colocalization of ingested opsonized apoptotic Jurkat and neutrophils with LTR.

Fig. 2.

Delayed phagosome acidification in dendritic cells. (A and B) Images showing low colocalization rates of carboxylate beads with LTR in JAWS2 (A) and HMDDC (B). (C and D) Colocalization with LTR and lysosensor in DC. (E) Rate of uptake expressed as phagocytic index.

Fig. 3.

Maturation of apoptotic cell phagsomes is Rho-dependent. (A and B) Effect of Rho inhibition on the colocalization of ingested targets with LTR in Mφ. Colocalization is significantly reduced by Y-27632 or C3T for phagosomes containing apoptotic cells or carboxylate beads but not for opsonized cells or amine beads. (C) Rho activity assay showing total and active Rho in J774. (D) No effect of Rho inhibition on colocalization of ingested cells with LTR in JAWS2 is seen.

Fig. 4.

ERM inhibition slows acidification of apoptotic cell phagosomes. (A) Transfected (N-terminal moesin) and untransfected fibroblasts that have ingested carboxylate beads. The successfully transfected cell (green fluorescence) does not stain for LTR, whereas the unsuccessfully transfected cells do. (B) Successful transfection significantly slows the rate of LTR colocalization of phagosomes containing carboxylate beads or apoptotic cells but not amine beads or opsonized cells. (C) Transfection with a control construct does not affect colocalization of ingested apoptotic neutrophils with LTR and no difference in colocalization between all Mφ (pink), transfected Mφ (light blue), and untransfected Mφ (purple).

Fig. 5.

Phagosome maturation and digestion. (A) (Left) Percentage of phagosomes containing apoptotic (blue) or opsonized neutrophils (orange) expressing Rab7 or LAMP-1 in J774 at 90 min. (Right) Images of J774 that have ingested PKH-labeled (green) apoptotic (Upper) or opsonized (Lower) neutrophils detected with a secondary red antibody against Rab 7. (B) (Left) Percentage of phagosomes containing either apoptotic (blue) or opsonized neutrophils (orange) expressing active cathepsin B, L, or K in J774 at 90 min. (Right) Images of J774 that have ingested PKH-labeled apoptotic (Upper) or opsonized (Lower) neutrophils stained for magic red cathepsin L.