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E.F. Nandrot, M. Anand, K. Atabai, S.C. Finnemann; The Integrin Ligand MFG–E8 Promotes RPE Phagocytosis of Photoreceptor Outer Segments in vitro and in vivo . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5880.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelial (RPE) cells use αvß5 integrin and Mer tyrosine kinase (MerTK) receptors to recognize and engulf shed photoreceptor outer segments (POS). Lack of αvß5 integrin receptors in mice abolishes rhythmic POS phagocytosis causing loss of vision and lipofuscin accumulation with age. Despite their potential importance in synchronizing POS uptake in the retina, ligands activating αvß5 integrin at the RPE apical surface are thus far unknown. Here we identify an important role for the soluble integrin ligand MFG–E8 in POS phagocytosis by RPE cells in vitro and in vivo.
We studied MFG–E8 mRNA and protein expression in rat RPE–J cells, whole retina and RPE tissues as well as its secretion by RPE in culture. We compared effects of wild–type and mutant recombinant MFG–E8 on uptake of isolated POS fragments by RPE cells in culture. We performed similar phagocytosis assays using unpassaged primary RPE cells isolated from MFG–E8 null and heterozygote mice. We compared MerTK phosphorylation in MFG–E8 null and heterozygote retina in vivo by quantitative immunoblotting.
We detected MFG–E8 transcript and protein in rat RPE–J cells, mouse retina and RPE. Moreover, RPE cells in culture secreted MFG–E8 constitutively and during in vitro phagocytosis. Addition of recombinant wild–type MFG–E8 to RPE cells in culture increased surface POS binding and engulfment. In contrast, recombinant mutant MFG–E8 lacking the RGD integrin recognition motif inhibited POS binding. Primary RPE cells isolated from MFG–E8 null mice showed reduced phagocytic activity compared to MFG–E8 heterozygote RPE cells despite normal epithelial morphology and apical surface localization of αvß5 integrin receptors. Strikingly, MFG–E8 null retina in vivo failed to stimulate peak MerTK phosphorylation that follows light onset in wild–type retina.
Increase of POS phagocytosis by recombinant MFG–E8 in vitro suggests that this integrin ligand promotes POS binding to αvß5 integrin receptors and subsequent activation of signaling pathways essential for particle engulfment. Furthermore, our in vivo results suggest that MFG–E8 may be solely responsible for the rhythmic daily signaling of αvß5 integrin to MerTK that synchronizes retinal phagocytosis. Studies are underway to fully elucidate the role of MFG–E8 in daily RPE phagocytosis and its regulation in the retina.
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