In recent years it has become apparent that removal of apoptotic cells (AC) by professional phagocytes alters the macrophage phenotype. This change is characterized by attenuated proinflammatory cytokine expression and NO production, which mechanistically remained unexplained. With the intention to explore molecular mechanisms underlying reduced NO formation, we showed that NO production in IFNgamma-stimulated murine RAW264.7 macrophages exposed to AC but not to either necrotic or viable human Jurkat cells was significantly reduced although iNOS expression remained high compared with controls. Analyzing iNOS activity in the cell extracts by using the radioactive L-arginine/citrulline conversion assay revealed increased ornithine production over citrulline in cells exposed to AC. RT-PCR, Western blot, and luciferase reporter analysis supported the idea of an arginase II increase in response to AC. The involvement of arginase in modulating NO formation in response to AC was substantiated by the arginase inhibitor N(omega)-hydroxy-nor-L-arginine. Moreover, knockdown of arginase II by siRNA allowed recovery of NO production. Experiments with AC-conditioned medium demonstrated that a soluble lipid factor, rather than phagocytosis of AC, modulated NO production in macrophages. We conclude that AC release a lipid factor to modulate NO formation in macrophages via arginase II up-regulation, thereby contributing to innate immune regulation.

Removal of apoptotic cells by phagocytes is considered a pivotal immune regulatory process. Although considerable knowledge has been obtained on the postphagocytic macrophage phenotype, there is little information on molecular mechanisms, which provoke macrophage polarization. In this study, we show that human apoptotic Jurkat cells (AC) or AC-conditioned medium (CM) rapidly induces cyclooxygenase-2 (COX-2) expression in mouse RAW264.7 macrophages via sphingosine-1-phosphate (S1P). Pharmacological inhibition of S1P release from AC or using CM from cells with a knockdown of sphingosine kinase 2 in human MCF-7 cells abrogates this effect. Expression of COX-2 resulted from an increase in mRNA stability via its 3′-untranslated region (UTR), shown by COX-2–3′-UTR and AU-rich element-driven reporter assays. Western analysis corroborated increased nucleocytoplasmic shuttling of the RNA-binding protein HuR after CM treatment. RNA EMSA analysis revealed an S1P- and CM-mediated increase in HuR-RNA binding to a COX-2-specific UTR, whereas HuR knockdown pointed to its importance for S1P in CM-induced COX-2 expression. Immunofluorescence microscopy of phospholipase A2 (PLA2) and ELISA analysis of PGE2 revealed activation of PLA2 and production of PGE2 in response to CM but not S1P. S1P, released from AC, uses HuR to stabilize COX-2 mRNA and thus to increase COX-2 protein expression. However, only CM also activates PLA2 to provide the substrate for COX-2. Our data underscore the importance of S1P in AC-mediated immune regulation, by stabilizing COX-2 mRNA in macrophages, a prerequisite for PGE2 formation.

Apoptotic cell (AC)-derived factors alter the physiology of macrophages (MΦs) towards a regulatory phenotype, characterized by reduced nitric oxide (NO) production. Impaired NO formation in response to AC-conditioned medium (CM) was facilitated by arginase II (ARG II) expression, which competes with inducible NO synthase for L-arginine. Here we explored signaling pathways allowing CM to upregulate ARG II in RAW264.7 MΦs. Sphingosine-1-phosphate (S1P) was required and acted synergistically with a so far unidentified factor to elicit high ARG II expression. S1P activated S1P(2), since S1P(2) knockdown prevented ARG II upregulation. Furthermore, ERK5 knockdown attenuated CM-mediated ARG II protein induction. CREB was implicated as shown by EMSA analysis and decoy-oligonucleotides scavenging CREB in RAW264.7 MΦs, which blocked ARG II expression. We conclude that AC-derived S1P binds to S1P(2) and acts synergistically with other factors to activate ERK5 and concomitantly CREB. This signaling cascade shapes an anti-inflammatory MΦ phenotype by ARG II induction.

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