How Does CD47-SIRPα ‘Don’t Eat Me Signal’ Physically Signal Self

Resident macrophages in spleen and liver are particularly adept at recognizing foreign pathogens through recognition of ‘non-self’ proteins on the pathogen surface but also through the absence of ‘self’ proteins that are highly displayed on circulating blood cells. Red blood cells display a ‘marker of self’ protein CD47 which increases the in vivo half-life and decreases red-pulp splenic macrophage uptake of mouse RBC (Oldenborg et al, Science 2000) and also of particles displaying human-CD47 in recent studies by our group (Rodriguez et al, Science 2013). CD47 signals self through its counter receptor SIRPa, which is highly expressed on the surfaces of myeloid cells but also highly polymorphic. The CD47 protein functions in vitro as a marker of self toward human SIRPa on human macrophages and monocytes, inhibiting accumulation of myosin II motor protein to the phagocytic synapse (Tsai 2008). The work here aims to clarify when and how CD47-SIRPa inhibition physically signals ‘self’ during macrophage phagocytosis uptake. While it is clear that CD47 reduces the number of uptake events, here we use time-lapse and confocal microscopy to examine the forces of distortion imparted by phagocytes on opsonized red blood cell targets during uptake. Glutaraldehyde-fixed RBC are also used as a model to assess the affects of cell rigidity in this self-recognition process, since rigidity is relevant to processes as diverse as RBC aging and sickle RBC to malaria but also because adhesion (by macrophages) is expected to activate the myosin-II contractility system and oppose CD47 signaling. Through blocking and pharmacological approaches, we parse the pathways between foreign, self, and rigidity sensing.