Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms

Stefan Walbaum,Benjamin Ambrosy,Paula Schütz,Anne C Bachg,Markus Horsthemke,Jeanette H.W Leusen,Attila Mócsai,Peter J Hanley

doi:10.1016/j.jbc.2021.100256

Abstract

A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and KO mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80+ cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2–3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcγRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10–25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.

Highlights

A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking
Kaplan deduced that mouse peritoneal macrophages engulfed immunoglobulin G (IgG)-opsonized sheep red blood cells “by means of thin membrane extensions rising from the macrophage surface and enclosing the opsonized particles tightly in a cup-like structure protruding from the macrophage surface” [5], in accord with the zipper model of phagocytosis [6]
Expression of Fcγ receptors (FcγRs) in mouse macrophages and real-time assay for complement-mediated hemolysis triggered by IgGopsonized human red blood cells (hRBCs)

Summary

Introduction

A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. These data show that the ingestion of IgG-opsonized hRBCs is mediated by phagocytic cup formation and requires the Fc receptor γ chain and a functional ITAM (Fig. 1, D and E), and sinking phagocytosis does not prevail in either Fcer1g−/− or NOTAM macrophages.

Results

Conclusion