Researchers question whether particles sink into cells

Formation of a phagocytic cup in a mouse macrophage during the uptake of human blood cells opsonised with complement C3b.
© Hanley, Liashkovich

Project titel: Cells Eating: is there a distinct complement-mediated sinking-in mode of phagocytosis?
Principal investigators: Peter J. Hanley, Ivan Liashkovich
Project time: 07/2016 - 10/2018
Project code: FF-2016-05

Phagocytosis is a receptor-mediated process in which immune cells internalize particles such as bacteria. In 1977, Gilla Kaplan identified two distinct pathways of phagocytosis: “reaching up”, initiated by Fcγ receptors, and “sinking in”-phagocytosis, mediated by complement receptors. In this CiM project, researchers are looking into whether a distinct “sinking in” process, mediated by complement receptors, actually exists.

In the case of “reaching up”-phagocytosis, a so-called phagocytic cup – a protrusion of the cell membrane along the particle – is formed. In the case of sinking in”-phagocytosis, in contrast, a particle is internalized without a cup being formed and appears to sink into the cell.

Dr. Peter Hanley, a physician and biologist, and Dr. Ivan Liashkovich, a biophysicist, are assuming that – in contrast to the conventional theory – both complement and Fcγ receptors can induce phagocytic cup formation.

Using spinning-disk confocal microscopy, the researchers generate high-resolution, time-lapse images in 3D in order to be able to examine the exact characteristics of the cup formed during the Fcγ receptor and complement receptor-mediated processes. In addition, the adhesive forces between receptor and particle are being measured by means of atomic force microscopy (AFM).

In their experiments, the researchers bring human red blood cells (erythrocytes) into contact with scavenger cells (macrophages) from a mouse. When blood cells are pre-coated (opsonised) with antibodies from a mouse, the researchers can observe Fcγ-receptor-mediated “reaching up”-phagocytosis. In comparison, no phagocytosis of these blood cells takes place in macrophages from transgenic mice in which Fcγ receptors are not functional. If, however, the erythrocytes are opsonised with the complement C3b, complement receptor-mediated “reaching up” phagocytosis can also be observed in the Fcγ mutant (see figure). The researchers want to carry out further experiments to show that, in both cases, “reaching up”-phagocytosis requires Syk-kinase.