Inhibitory immunoreceptors CD300a and CD300lf cooperate to regulate mast cell activation

Mast cells (MCs) play a central role in allergic immune responses. MC activation is regulated by several inhibitory immunoreceptors. The CD300 family members CD300a and CD300lf recognize phospholipid ligands and inhibit the FcεRI-mediated activating signal in MCs. While CD300a binds to phosphatidylserine (PS) to inhibit MCs activation, CD300lf function is less clear due to its ability to bind with ceramide and PS. Moreover, it also remains blurring whether CD300a and CD300lf function independently, cooperatively, or by interfering with each other in regulating MC activation. Using imaging and flow cytometric analyses of bone marrow-derived cultured MCs (BMMCs) from wild-type (WT), Cd300a-/-, Cd300lf-/-, and Cd300a-/-Cd300lf-/- mice, we show that CD300lf and CD300a colocalized with PS externalized to the outer leaflet of the plasma membrane with a polar formation upon activation, and CD300lf cooperates with CD300a to inhibit BMMCs activation. CD300lf also colocalized with extracellular ceramide in addition to the internal PS on the cell surface, which results in stronger inhibition of MC activation than CD300lf binding to PS alone. Similarly, although both Cd300a-/- and Cd300lf-/- mice showed decreased rectal temperatures compared with WT mice in the model of passive systemic anaphylaxis, Cd300a-/-Cd300lf-/- mice showed lower rectal temperature than either Cd300a-/- or Cd300lf-/- mice. Our results demonstrate the cooperativity of multiple inhibitory receptors expressed on MCs and their regulatory functions upon binding to respective ligands.