We identified and characterized the function of mouse CD300f. We showed that CD300f-mediated phagocytosis of apoptotic cells (AC )occurs in a phosphatidylserine (PS) dependent manner, as annexin V, MFG-E8 or PS-containing liposomes block CD300f-mediated phagocytosis. Additionally, cells expressing CD300f phagocytize beads coated with PS liposomes. It is important to note that a membrane environment is most likely critical for CD300f binding to PS, as it has been difficult to detect CD300f binding to PS, which is extraneous of liposomes. Even though phosphatidylglycerol can also be recognized by CD300f, the facts that PG is not commonly found in eukaryotic plasma membranes and that MFG-E8, which is PS specific,completely blocks CD300f-mediated phagocytosis rule out a significant role for PG in CD300f-mediated phagocytosis of AC. CD300f was recently reported to bind ceramide. However, we found that liposomes containing ceramide failed to block CD300f-mediated phagocytosis, indicating that, in contrast to PS, ceramide does not play an important role in the function of CD300f as a molecule mediating recognition and engulfment of AC. Impaired AC clearance is often associated with the development of autoimmunity, as is seen in mice deficient in expression of several PS-recognition proteins. Surprisingly, C57BL/6 mice deficient in CD300f are relatively healthy, which may be due to their genetic background and the redundancy in PS recognition receptors. For instance, MFG-E8-deficient mice produce autoantibodies on a mixed 129/B6 background but not on a C57BL/6 genetic background. In addition, mice deficient in TIM4 develop little or no autoantibodies on C57BL/6 background, unless the mice are also deficient in MFG-E8. Similarly, we found that mice deficient in both FcgRIIB, which predisposes to systemic lupus erythematosus (SLE), and CD300f exhibit an accelerated and more lethal course of autoimmune disease, associated with splenomegaly, autoantibody production and tissue pathology. Thus, the impaired clearance of AC associated with CD300f deficiency exacerbates the development of autoimmunity. CD300f expression clearly promotes AC phagocytosis by primary macrophages and cell lines, in a manner that is largely dependent on the capacity of CD300f to signal. We showed that AC recognition strongly enhances the phosphorylation of CD300f tyrosines, predominantly on Y276, which leads to p85a recruitment to CD300f. This is followed by activation of PI3K, as demonstrated by the production of PI(3,4,5)P3 and Akt phosphorylation. The phosphorylation of the other four tyrosines leads to inhibition of phagocytosis by inhibiting PI3K activation, as evidenced by changes in Akt phosphorylation. A mutation of any one of the four tyrosines (Y241, Y289, Y303 and Y325) resulted in the increased AC phagocytosis, indicating that those tyrosine-based motifs are indeed involved in the initiation of an inhibitory signal. Most likely, SHP1 is the phosphatase involved in the inhibitory signaling, as it associates with CD300f in J774 and HeLa cells, and SHP1 overexpression decreases CD300f-mediated AC phagocytosis in a dose-dependent manner. In agreement, SHP1 has been shown to be recruited to Fc receptor-mediated phagosomes and negatively regulate phagocytosis. The maximal association between CD300f and SHP1 has been reported to require several tyrosines (Y241, Y289 and Y325). Exactly how the multiple tyrosines cooperatively associate with SHP1 for the regulation of phagocytosis needs further study. Intriguingly, mouse CD300f has been shown to serve as an activating or inhibitory receptor. Our data indicate that CD300f might function differently depending on the signaling capacity available within a cell type. In fibroblast cells, expression of CD300f WT generates positive signals resulting from phosphorylation of Y276 that dominate over negative signals. In J774 macrophages, however, the inhibitory cues appear to override the positive signal, as cells expressing CD300f WT or Y276F mutant showed reduced phagocytic activity compared with cells expressing signaling-deficient forms. A possible explanation could be that SHP1 is expressed at much higher levels in J774 cells than in L929 cells, which may lead to its more efficient recruitment by CD300f resulting in reduced PI3K activation. Alternatively, the inositol 5-phosphatase, SHIP1, expressed in J774 but not in L929 cells, could hydrolyse PI(3,4,5)P3 resulting in deactivation of PI3K. In this regard, SHIP1 was reported to be recruited to phagocytic cups and regulate phagocytosis of IgG-opsonized particles. Even though we and others did not detect an association of SHIP1 with CD300f, the phosphatase might be recruited to phagocytic cups by other, unknown receptors, and thus indirectly decrease the CD300f-initiated activation of PI3K in macrophage cells. Likewise, the recruitment of SHP1 by CD300f could down modulate positive signals for phagocytosis generated by other receptors. Thus, the availability of phosphatases could affect the functions played by CD300f in a particular cell type. PI3K kinase activity appears to be essential for phagocytosis, as PI3K inhibitors impair target engulfment for both Fc receptor- and PS-recognition receptor-mediated phagocytosis. Local activation of PI3K is required to induce actin reorganization and subsequent uptake of IgG-opsonized targets, presumably related to the PI3K requirement for pseudopodia extension and generation of contractile force on phagosomes by the recruitment of myosin X. In this study, we present the details about CD300fs function in AC engulfment,highlighting the involvement of PI3K and actin rearrangements. We propose that CD300f accumulates at AC contact sites that will evolve into the invaginating part of phagocytic cups and where following phosphorylation in response to PS recognition, it recruits p85a and activates PI3K. This, in turn, results in PI3K phosphorylating PI(4,5)P2 to PI(3,4,5)P3. As PI(4,5)P2 associates with gelsolin, profilin and other proteins regulating actin cytoskeleton, the absence of PI(4,5)P2 could lead to the clearance of cortical F-actin from the invaginating part of the cup during CD300f-mediated phagocytosis. This would eliminate a possible physical barrier created by the actin meshwork and decrease the membrane rigidity, thereby allowing for easier membrane deformation and AC internalization. At the same time, the increase in PI(3,4,5)P3 levels would serve to activate the small GTPase Rac/Cdc42 pathway to promote F-actin assembly along the protruding part of the phagocytic cups for membrane extension, required for phagocytic cup closure. The fact that latrunculin treatment blocks CD300f-mediated protrusion extension and phagosome sealing without interruption of membrane cup formation supports the notion that F-actin is unnecessary for the initial membrane cup formation, but is required for further cup extension and phagosome sealing. In summary, our data demonstrates that CD300f recognizes PS exposed on the AC surface, and transduces signals for PI3K activation and F-actin remodeling, thereby promoting AC phagocytosis. Consequently, a deficiency in CD300f leads to impaired AC clearance and exacerbates the development of autoimmune disease.
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