Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin β3. We have previously shown that TG2−/− mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin β3, a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin β3 to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin β3 and Rac1. In the absence of TG2, integrin β3 cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals.

In the human body, billions of cells die by apoptosis every day. The subsequent clearance of apoptotic cells by phagocytosis is normally efficient enough to prevent secondary necrosis and the consequent release of cell contents that would induce inflammation and trigger autoimmunity. In addition, apoptotic cells generally induce an anti-inflammatory response, thus removal of apoptotic cells is usually immunologically silent. Since the first discovery that uptake of apoptotic cells leads to transforming growth factor (TGF)-β and interleukin (IL)-10 release by engulfing macrophages, numerous anti-inflammatory mechanisms triggered by apoptotic cells have been discovered, including release of anti-inflammatory molecules from the apoptotic cells, triggering immediate anti-inflammatory signaling pathways by apoptotic cell surface molecules via phagocyte receptors, activating phagocyte nuclear receptors following uptake and inducing the production of anti-inflammatory soluble mediators by phagocytes that may act via paracrine or autocrine mechanisms to amplify and preserve the anti-inflammatory state. Here, we summarize our present knowledge about how these anti-inflammatory mechanisms operate during the clearance of apoptotic cells.

In healthy individuals, billions of cells die by apoptosis every day. Removal of the dead cells by phagocytosis (a process called efferocytosis) must be efficient to prevent secondary necrosis and the consequent release of pro-inflammatory cell contents that damages the tissue environment and provokes autoimmunity. In addition, detection and removal of apoptotic cells generally induces an anti-inflammatory response. As a consequence improper clearance of apoptotic cells, being the result of either genetic anomalies and/or a persistent disease state, contributes to the establishment and progression of a number of human chronic inflammatory diseases such as autoimmune and neurological disorders, inflammatory lung diseases, obesity, type 2 diabetes, or atherosclerosis. During the past decade, our knowledge about the mechanism of efferocytosis has significantly increased, providing therapeutic targets through which impaired phagocytosis of apoptotic cells and the consequent inflammation could be influenced in these diseases.

Previous work in our laboratory has shown that transglutaminase 2 (TG2) acting as a coreceptor for integrin β3 is required for proper phagocytosis of apoptotic cells. In the absence of TG2, systemic lupus erythematosus–like autoimmunity develops in mice, similarly to other mice characterized by a deficiency in the clearance of apoptotic cells. In this study, we demonstrate that increasing TG2 expression alone in wild-type macrophages is not sufficient to enhance engulfment. However, during engulfment, the lipid content of the apoptotic cells triggers the lipid-sensing receptor liver X receptor (LXR), which in response upregulates the expression of the phagocytic receptor Mer tyrosine kinase and the phagocytosis-related ABCA1, and that of retinaldehyde dehydrogenases leading to the synthesis of a nonclassical retinoid. Based on our retinoid analysis, this compound might be a dihydro-retinoic acid derivative. The novel retinoid then contributes to the upregulation of further phagocytic receptors including TG2 by ligating retinoic acid receptors. Inhibition of retinoid synthesis prevents the enhanced phagocytic uptake induced by LXR ligation. Our data indicate that stimulation of LXR enhances the engulfment of apoptotic cells via regulating directly and indirectly the expression of a range of phagocytosis-related molecules, and its signaling pathway involves the synthesis of a nonclassical retinoid. We propose that retinoids could be used for enhancing the phagocytic capacity of macrophages in diseases such as systemic lupus erythematosus, where impaired phagocytosis of apoptotic cells plays a role in the pathogenesis of the disease.

The thymus provides the microenvironment in which thymocytes develop into mature T cells, and interactions with thymic stromal cells are thought to provide the necessary signals for thymocyte maturation. Recognition of self-MHC by T cells is a basic requirement for mature T-cell functions, and those thymocytes that do not recognize the peptide-loaded self-MHC molecules found in the thymus, and therefore lack a TCR signal, undergo a default death pathway named "death by neglect" in the thymic cortex. In the absence of this TCR signaling, it has been suggested that binding of glucocorticoids toor the ligation of certain cell surface molecules, such as CD8, CD24, CD45, or CD99 on -these neglected thymocytes will induce them to enter the apoptotic program. Apoptotic thymocytes are cleared by the surrounding macrophages and, as a consequence, these macrophages are known to release various molecules, such as adenosine, retinoids, TGF-β, ATP, and carbon monoxide. Interestingly, all these molecules have been described to induce or promote apoptosis in thymocytes in the absence of TCR signaling. Here, we propose that thymic macrophages, because they continually engulf apoptotic cells, might constantly provide these cell death-inducing signals, and thus contribute to the formation of a thymic milieu that ensures the effective induction of "death by neglect".