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.

TNF-α, a potent proinflammatory cytokine, is generated in a precursor form called transmembrane (m)TNF-α that is expressed as a type II polypeptide on the surface of certain cells. mTNF-α was shown to act both as a ligand by binding to TNF-α receptors, as well as a receptor that transmits outside-to-inside (reverse) signals back into the mTNF-α–bearing cells. In this study, we show that nonactivated macrophages express basal levels of mTNF-α and respond to anti–TNF-α Abs by triggering the MAPK kinase 4 signaling pathway. The pathway induces TGF-β. Based on inhibitory experiments, the production of TGF-β1 is regulated via Jun kinases, whereas that of other TGF-βs is regulated via p38 MAPKs. Exposure to LPS further induced the expression of mTNF-α, and triggering of mTNF-α strongly suppressed the LPS-induced proinflammatory response. Neutralizing TGF-β by Abs prevented the mTNF-α–mediated suppression of LPS-induced proinflammatory cytokine formation, indicating that the immune-suppressive effect of mTNF-α is mediated via TGF-β. Although apoptotic cells are also known to suppress LPS-induced proinflammatory cytokine formation in macrophages by upregulating TGF-β, we show that they do not use the mTNF-α signaling pathway. Because TGF-β possesses a wide range of immune-suppressive effects, our data indicate that upregulation of TGF-β synthesis by those TNF-α–targeting molecules, which are able to trigger mTNF-α, might contribute to their therapeutic effect in the treatment of certain inflammatory diseases such as Crohn’s disease, Wegener’s granulomatosis, or sarcoidosis. Additionally, none of the TNF-α–targeting molecules is expected to interfere with the immune-silencing effects of apoptotic cells.

Transglutaminase 2 (TG2) has been known for a long time to be associated with the in vivo apoptosis program of various cell types including T cells. Though the expression of the enzyme was strongly induced in mouse thymocytes following apoptosis induction in vivo, no significant induction of TG2 could be detected, when thymocytes were induced to die by the same stimuli in vitro indicating that signals arriving from the tissue environment are required for the in vivo induction of the enzyme in apoptotic thymocytes. Previous studies have shown that one of these signals is transforming growth factor-β (TGF-β) which is released by macrophages engulfing apoptotic cells. Besides TGF-β, the TG2 promoter contains retinoic acid response elements as well. Here we show that in vitro retinoic acids, or TGF-β and retinoic acids together can significantly enhance the TG2 mRNA expression in dying thymocytes, and the apoptotic signal contributes to the TG2 induction. Inhibition of retinoic acid synthesis either by alcohol or retinaldehyde dehydrogenases significantly attenuates the in vivo induction of TG2 following apoptosis induction indicating that retinoids indeed might contribute in vivo to the apoptosis-related TG2 expression. What is more, the in vivo apoptosis induction in the thymus is accompanied by an enhanced retinoid-dependent transcriptional activity due to the enhanced retinoid synthesis by macrophages engulfing apoptotic cells. Our data reveal a new crosstalk between macrophages and apoptotic cells, in which apoptotic cell uptake-induced retinoid synthesis in macrophages enhances TG2 expression in the dying thymocytes.

Adenosine is known to reduce inflammation by suppressing the activity of most immune cells. Previous studies have shown that lipopolysaccharide (LPS) stimulated mouse macrophages produce adenosine, and the adenosine A2A receptor (A2AR) signaling activated in an autocrine manner attenuates LPS-induced pro-inflammatory cytokine formation. It has been suggested that A2AR signaling inhibits LPS-induced pro-inflammatory cytokine production through a unique cAMP-dependent, but PKA- and Epac-independent signaling pathway. However, the mechanism of inhibition was not identified so far. Here we report that LPS stimulation enhances A2AR expression in mouse bone marrow derived macrophages, and loss of A2ARs results in enhanced LPS-induced pro-inflammatory response. Loss of A2ARs in A2AR null macrophages did not alter the LPS-induced NF-κB activation, but an enhanced basal and LPS-induced phosphorylation of MAP kinases (especially that of JNKs) was detected in A2AR null cells. A2AR signaling did not alter the LPS-induced phosphorylation of their upstream kinases, but by regulating adenylate cyclase activity it enhanced the expression of dual specific phosphatase (DUSP)1, a negative regulator of MAP kinases. As a result, lower basal and LPS-induced DUSP1 mRNA and protein levels can be detected in A2AR null macrophages. Silencing of DUSP1 mRNA expression resulted in higher basal and LPS-induced JNK phosphorylation and LPS-induced pro-inflammatory cytokine formation in wild type macrophages, but had no effect on that in A2AR null cells. Our data indicate that A2AR signaling regulates both basal and LPS-induced DUSP1 levels in macrophages via activating the adenylate cyclase pathway.

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