Ceramide-1-Phosphate a Novel Mediator for Phagocytosis.

We identified the generation of ceramide-1-phosphate (C1P) through activation of a ceramide kinase in neutrophils. Our previous studies indicated that C1P enhanced calcium-dependent fusion of liposomes. We hypothesized that human ceramide kinase (hCERK) activity and C1P synthesis leads to enhanced phagocytosis through a mechanism involving modulating membrane fluidity and Ca²⁺ generation. hCERK was stably transfected into COS-1 cells bearing FcγRIIA. hCERK activity was 2.3 times higher in cells transfected with hCERK than in the FcγRIIA cells or cells transfected with pc-vector. Stably transfected cells showed a 3-fold increase in phagocytosis. Besides increasing phagocytosis, the percentage of ingesting COS-1 cells increased from 43+ 11 in control cells and 50 + 11 in pc-vector control to 70 + 9 (p<0.0001, n=6) in pc-hCERK transfected cells. Cells labeled with [³H]-D-erythro-sphingosine and challenged with particles increased both phagocytosis by three fold and C1P levels by two times compared to resting controls. FcγRIIA, pc-vector and pc-hCERK transfected cells were subjected to cellular fractionation. Utilizing an antibody to c-Myc we confirmed that c-Myc tagged pc-hCERK was localized in the raft fraction, which was identified by a caveolin-1 marker. To assess plasma membrane fluidity we labeled COS–1 cells with 2-dimethylamino-6-lauroylnaphthalene (Laurdan). Cells transfected with pc-hCERK showed higher liquid crystalline order than control and vector transfected cells, a condition favorable to promote membrane fusion. Such ordered structures are reported to be the site of Ca²⁺ waves ignition. High speed imaging revealed that cells bearing pc-hCERK showed two Ca²⁺ waves beginning at the leading edge of the cell that propagated in both directions. When the two waves reached the vicinity of the phagosome, a secondary waves split off from each of them, then propagated about the perimeter of the phagosome. That was inhibitable by employing a store-operated Ca²⁺ channel (SOC) inhibitor. This behavior is unique to the FcγRIIA/pc-hCERK transfected cells. In conclusion transfected COS-1 cells were able to increase their C1P levels during phagocytosis. This changed the structural order parameter of the lipid rafts where hCERK is localized and likely contributed to phagocytosis by promoting phagosome development. Lipid rafts were enriched in Ca²⁺ signaling machinery and in turn pc-hCERK transfection resulted in a novel means to markedly enhance phagocytosis by generating Ca²⁺ movement from SOC.