While much is known about the macrophage receptors important for the
phagocytosis of apoptotic cells, little is known about how macrophages first
"sense" and then move toward dying cells. Work presented here investigates the
chemoattractant molecules released by dying cells in the "Come-get-me" stage
of apoptotic cell clearance.
The CD 14 knockout mouse (CD 14-/-) was observed to have increased
numbers of free apoptotic cells in the thymus. It was hypothesised that this was
due to a defect in macrophage clearance of dying cells. In vitro assays revealed
that macrophages from the CD 14-/- mouse had a partial defect in the
phagocytosis of apoptotic cells. Clearance also involves macrophage movement
towards the apoptotic corpse. Therefore, it was hypothesised that, in addition to
a phagocytosis defect, CD 14-/- had impaired chemotaxis to apoptotic cells.
An in vitro transmigration assay was developed using the Burkitt's
lymphoma (BL) cells as a source of apoptotic cells. In this assay, monocytes
and macrophages, but not neutrophils, migrated preferentially to apoptotic BL,
and chemotaxis correlated to phosphatidylserine exposure on apoptotic cells.
Chemotaxis was abolished when BL were transfected with the anti-apoptotic
protein bcl-2. Although CD 14 was up regulated on the surface of the migrating
macrophages, experiments with knockout cells revealed that chemotaxis did not
require either CD 14 or the scavenger receptor CD36.
Experiments using a viral chemokine antagonist (vMIPII) suggested that
fractalkine (CX3CL1) was a candidate "come-get-me" signal released from
dying cells. Fractalkine was expressed by apoptotic cells and in chemotaxis
assays, CX3CL1 competitively inhibited macrophage migration to apoptotic
cells but not to CCL5 (RANTES). Monoclonal antibodies that blocked the
chemokine domain of CX3CL1 also inhibited macrophage chemotaxis to
This is the first demonstration of the involvement of a known chemokine,
CX3CL1 in the clearance of apoptotic cells. In this novel process, cells dying by
apoptosis release fractalkine that recruits macrophages primed to efficiently
engulf the cell corpse.