Phagocytosis of apoptotic cells (efferocytosis) induces macrophage differentiation towards a regulatory phenotype (IL-10high/IL-12p40low). CD36 is involved in the recognition of apoptotic cells (AC), and we have shown that the platelet-activating factor receptor (PAFR) is also involved. Here, we investigated the contribution of PAFR and CD36 to efferocytosis and to the establishment of a regulatory macrophage phenotype. Mice bone marrow-derived macrophages were cocultured with apoptotic thymocytes, and the phagocytic index was determined. Blockage of PAFR with antagonists or CD36 with specific antibodies inhibited the phagocytosis of AC (~70–80%). Using immunoprecipitation and confocal microscopy, we showed that efferocytosis increased the CD36 and PAFR colocalisation in the macrophage plasma membrane; PAFR and CD36 coimmunoprecipitated with flotillin-1, a constitutive lipid raft protein, and disruption of these membrane microdomains by methyl-β-cyclodextrin reduced AC phagocytosis. Efferocytosis induced a pattern of cytokine production, IL-10high/IL-12p40low, that is, characteristic of a regulatory phenotype. LPS potentiated the efferocytosis-induced production of IL-10, and this was prevented by blocking PAFR or CD36. It can be concluded that phagocytosis of apoptotic cells engages CD36 and PAFR, possibly in lipid rafts, and this is required for optimal efferocytosis and the establishment of the macrophage regulatory phenotype. 1. Introduction Clearance of apoptotic cells (AC) by macrophages, also called efferocytosis, plays a central role in tissue homeostasis, and the impaired clearance of altered cells has been associated with the development of autoimmune and chronic inflammatory diseases [1]. Macrophages can acquire distinct phenotypes depending on the stimulus. M1 or classically activated macrophages are induced by the recognition of PAMPs (pathogen associated molecular patterns) and by proinflammatory cytokines. These cells exhibit high microbicidal activity and induce inflammation. In contrast, M2 macrophages produce anti-inflammatory and tissue remodelling cytokines and can be divided into subtypes according to the stimulus; the alternatively activated macrophages are induced by Th2 cytokines, and the regulatory macrophages are induced by anti-inflammatory cytokines, immune complexes, apoptotic cells, and oxidised lipids, among other stimuli [2, 3]. The first evidence that efferocytosis induces a suppressor phenotype came from the studies of Fadok et al. [4]. The authors showed that the addition of apoptotic cells to macrophages inhibited the
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