p85α recruitment by the CD300f phosphatidylserine receptor mediates apoptotic cell clearance required for autoimmunity suppression

Abstract

Apoptotic cell (AC) clearance is essential for immune homeostasis. Here we show that mouse CD300f (CLM-1) recognizes outer membrane-exposed phosphatidylserine, and regulates the phagocytosis of ACs. CD300f accumulates in phagocytic cups at AC contact sites. Phosphorylation within CD300f cytoplasmic tail tyrosine-based motifs initiates signals that positively or negatively regulate AC phagocytosis. Y276 phosphorylation is necessary for enhanced CD300f-mediated phagocytosis through the recruitment of the p85α regulatory subunit of phosphatidylinositol-3-kinase (PI3K). CD300f-PI3K association leads to activation of downstream Rac/Cdc42 GTPase and mediates changes of F-actin that drive AC engulfment. Importantly, primary macrophages from CD300f-deficient mice have impaired phagocytosis of ACs. The biological consequence of CD300f deficiency is predisposition to autoimmune disease development, as FcγRIIB-deficient mice develop a systemic lupus erythematosus-like disease at a markedly accelerated rate if CD300f is absent. In this report we identify the mechanism and role of CD300f in AC phagocytosis and maintenance of immune homeostasis.

CD300b regulates the phagocytosis of apoptotic cells via phosphatidylserine recognition

“…To address directly whether CD300b recognizes TIM1, the interaction between TIM1 and CD300b-Fc was analyzed by SPR in the presence or absence of liposomes. Although the association level of CD300b-Fc to TIM1 was similar to reactions containing the Figure 3b; and Tian et al 19 ). Microscopic ( Figure 4a) and flow cytometric (Figure 4b) analyses demonstrated that CD300b-DAP12-expressing L929 cells efficiently engulfed PS-coated liposome beads, whereas CD300b-transduced cells showed association but not engulfment of PS-containing beads (Figures 4a and b).…”

“…33 The CD300 receptors are another such family expressed mainly on myeloid cells. Furthermore, work from us and others showed that at least three members of this family, CD300a, 16,26 CD300f, 18,19 and now CD300b, recognize PS and positively or negatively regulate the phagocytosis of apoptotic cells. CD300a and CD300f have been reported to negatively regulate phagocytosis, 17,19 which correlates with the fact that they contain ITIM motifs in their cytoplasmic tails that can bind phosphatases.…”

“…In this study, we characterize PS as a ligand of CD300b by utilizing phospholipid-coated liposomes, an approach successfully used in identifying ligands for other CD300 family members. 18,19 The previous studies failed to detect PS binding when using flat surfaces, which could restrict ligand access, an experimental limitation previously shown for plates coated with phospholipids and membrane spotted phospholipids. 24,25 Thus, the ligand binding by CD300 family members most likely depends on the presence or mimicking of the physiological cell surface environment.…”

“…We analyzed the binding of recombinant murine CD300b to phospholipids known to associate with CD300 molecules [16][17][18][19][24][25][26] by surface plasmon resonance (SPR), and found that CD300b-Fc bound to phosphatidylserine (PS)-containing liposomes, but not those containing phosphatidylcholine (PC), phosphatidylethanolamine (PE) or ceramide ( Figure 1a; Supplementary Figure 1). A control protein, NITR-Fc, 24 failed to bind to any liposomes (Figure 1b).…”

“…2 Multiple receptors for PS exist on phagocytic cells, although not necessarily simultaneously; these include stabilins, 9,10 T cell Ig mucin (TIM) 1 and TIM4, 11,12 BAI1,13 MFGE8, which bridges PS to integrin avb3, 14 and Protein S and Gas6, which bridge PS to TAM receptors. 15 Recently, we and others demonstrated that the CD300 family members, human and mouse CD300a, 16,17 and mouse CD300f, 18,19 also bind PS, and their expression regulates apoptotic cell phagocytosis.…”