Poly(ADP-ribosyl)ation of high mobility group box 1 (HMGB1) protein enhances inhibition of efferocytosis

Phagocytosis of apoptotic cells by macrophages, known as efferocytosis, is a critical process in the resolution of inflammation. High mobility group box 1 (HMGB1) protein was first described as a nuclear nonhistone DNA-binding protein, but is now known to be secreted by activated cells during inflammatory processes, where it participates in diminishing efferocytosis. Although HMGB1 is known to undergo modification when secreted, the effect of such modifications on the inhibitory actions of HMGB1 during efferocytosis have not been reported. In the present studies, we found that HMGB1 secreted by Toll-like receptor 4 (TLR4) stimulated cells is highly poly(ADP-ribosyl)ated (PARylated). Gene deletion of poly(ADP)-ribose polymerase (PARP)-1 or pharmacological inhibition of PARP-1 decreased the release of HMGB1 from the nucleus to the extracellular milieu after TLR4 engagement. Preincubation of macrophages or apoptotic cells with HMGB1 diminished efferocytosis through mechanisms involving binding of HMGB1 to phosphatidylserine on apoptotic cells and to the receptor for advanced glycation end products (RAGE) on macrophages. Preincubation of either macrophages or apoptotic cells with PARylated HMGB1 inhibited efferocytosis to a greater degree than exposure to unmodified HMGB1, and PARylated HMGB1 demonstrated higher affinity for phosphatidylserine and RAGE than unmodified HMGB1. PARylated HMGB1 had a greater inhibitory effect on Ras-related C3 botulinum toxin substrate 1 (Rac-1) activation in macrophages during the uptake of apoptotic cells than unmodified HMGB1. The present results, showing that PARylation of HMGB1 enhances its ability to inhibit efferocytosis, provide a novel mechanism by which PARP-1 may promote inflammation.     

Ligand-dependent kinase activity of MERTK drives efferocytosis in human iPSC-derived macrophages

Removal of apoptotic cells by phagocytes (also called efferocytosis) is a crucial process for tissue homeostasis. Professional phagocytes express a plethora of surface receptors enabling them to sense and engulf apoptotic cells, thus avoiding persistence of dead cells and cellular debris and their consequent effects. Dysregulation of efferocytosis is thought to lead to secondary necrosis and associated inflammation and immune activation. Efferocytosis in primarily murine macrophages and dendritic cells has been shown to require TAM RTKs, with MERTK and AXL being critical for clearance of apoptotic cells. The functional role of human orthologs, especially the exact contribution of each individual receptor is less well studied. Here we show that human macrophages differentiated in vitro from iPSC-derived precursor cells express both AXL and MERTK and engulf apoptotic cells. TAM RTK agonism by the natural ligand growth-arrest specific 6 (GAS6) significantly enhanced such efferocytosis. Using a newly-developed mouse model of kinase-dead MERTK, we demonstrate that MERTK kinase activity is essential for efferocytosis in peritoneal macrophages in vivo. Moreover, human iPSC-derived macrophages treated in vitro with blocking antibodies or small molecule inhibitors recapitulated this observation. Hence, our results highlight a conserved MERTK function between mice and humans, and the critical role of its kinase activity in homeostatic efferocytosis.Subject terms: Immune cell death, Peritoneal macrophages     

Neuroendocrine Signaling Via the Serotonin Transporter Regulates Clearance of Apoptotic Cells

Serotonin (5-hydroxytryptamine; 5-HT) is a CNS neurotransmitter increasingly recognized to exert immunomodulatory effects outside the CNS that contribute to the pathogenesis of autoimmune and chronic inflammatory diseases. 5-HT signals to activate the RhoA/Rho kinase (ROCK) pathway, a pathway known for its ability to regulate phagocytosis. The clearance of apoptotic cells (i.e. efferocytosis) is a key modulator of the immune response that is inhibited by the RhoA/ROCK pathway. Because efferocytosis is defective in many of the same illnesses where 5-HT has been implicated in disease pathogenesis, we hypothesized that 5-HT would suppress efferocytosis via activation of RhoA/ROCK. The effect of 5-HT on efferocytosis was examined in murine peritoneal and human alveolar macrophages, and its mechanisms were investigated using pharmacologic blockade and genetic deletion. 5-HT impaired efferocytosis by murine peritoneal macrophages and human alveolar macrophages. 5-HT increased phosphorylation of myosin phosphatase subunit 1 (Mypt-1), a known ROCK target, and inhibitors of RhoA and ROCK reversed the suppressive effect of 5-HT on efferocytosis. Peritoneal macrophages expressed the 5-HT transporter and 5-HT receptors (R) 2a, 2b, but not 2c. Inhibition of 5-HTR2a and 5-HTR2b had no effect on efferocytosis, but blockade of the 5-HT transporter prevented 5-HT-impaired efferocytosis. Genetic deletion of the 5-HT transporter inhibited 5-HT uptake into peritoneal macrophages, prevented 5-HT-induced phosphorylation of Mypt-1, reversed the inhibitory effect of 5-HT on efferocytosis, and decreased cellular peritoneal inflammation. These results suggest a novel mechanism by which 5-HT might disrupt efferocytosis and contribute to the pathogenesis of autoimmune and chronic inflammatory diseases.     

Monocyte chemoattractant protein-1/CC chemokine ligand 2 enhances apoptotic cell removal by macrophages through Rac1 activation

Apoptotic cell removal (efferocytosis) is an essential process in the regulation of inflammation and tissue repair. We have shown that monocyte chemoattractant protein-1/CC chemokine ligand 2 (MCP-1/CCL2) enhances efferocytosis by alveolar macrophages in murine bacterial pneumonia. However, the mechanism by which MCP-1 exerts this effect remains to be determined. Here we explored that hypothesis that MCP-1 enhances efferocytosis through a Rac1/phosphatidylinositol 3-kinase (PI3-kinase)-dependent mechanism.We assessed phagocytosis of apoptotic cells by MCP-1 treated murine macrophages in vitro and in vivo. Rac activity in macrophages was measured using a Rac pull down assay and an ELISA based assay (GLISA). The downstream Rac1 activation pathway was studied using a specific Rac1 inhibitor and PI3-kinase inhibitor in in vitro assays.MCP-1 enhanced efferocytosis of apoptotic cells by murine alveolar macrophages (AMs), peritoneal macrophages (PMs), the J774 macrophage cell line (J774s) in vitro, and murine AMs in vivo. Rac1 activation was demonstrated in these cell lines. The effect of MCP-1 on efferocytosis was completely negated by the Rac1 inhibitor and PI3-kinase inhibitor.We demonstrated that MCP-1 enhances efferocytosis in a Rac1-PI3 kinase-dependent manner. Therefore, MCP-1-Rac1-PI3K interaction plays a critical role in resolution of acute lung inflammation.     

Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis

Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. Bone marrow transplantation studies in ApoE- and LDLR-deficient mice revealed that hematopoietic scavenger receptor class B type I (SR-BI) deficiency results in severely defective efferocytosis in mouse atherosclerotic lesions, resulting in a 17-fold higher ratio of free to macrophage-associated dead cells in lesions containing SR-BI^−/− cells, 5-fold more necrosis, 65.2% less lesional collagen content, nearly 7-fold higher dead cell accumulation, and 2-fold larger lesion area. Hematopoietic SR-BI deletion elicited a maladaptive inflammatory response [higher interleukin (IL)-1β, IL-6, and TNF-α lower IL-10 and transforming growth factor β]. Efferocytosis of apoptotic thymocytes was reduced by 64% in SR-BI^−/− versus WT macrophages, both in vitro and in vivo. In response to apoptotic cells, macrophage SR-BI bound with phosphatidylserine and induced Src phosphorylation and cell membrane recruitment, which led to downstream activation of phosphoinositide 3-kinase (PI3K) and Ras-related C3 botulinum toxin substrate 1 (Rac1) for engulfment and clearance of apoptotic cells, as inhibition of Src decreased PI3K, Rac1-GTP, and efferocytosis in WT cells. Pharmacological inhibition of Rac1 reduced macrophage efferocytosis in a SR-BI-dependent fashion, and activation of Rac1 corrected the defective efferocytosis in SR-BI^−/− macrophages. Thus, deficiency of macrophage SR-BI promotes defective efferocytosis signaling via the Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation.     

Close Encounters of the First Kind: Innate Sensors and Multiple Sclerosis

Although autoimmune diseases by definition imply adaptive immune system pathologies, growing evidence points to the relevance of innate receptors in modulating the initiation and progression of the autoreactive response. Multiple sclerosis (MS) is a chronic autoimmune disease characterised by central nervous system (CNS) demyelination, inflammation and axonal damage, in which the role of several pathogens such as herpes viruses have long been described as potential triggers. Encounters of these pathogens with altered innate receptors in susceptible individuals might drive pathological autoreactivity and inflammation, overcoming tolerance and causing subsequent CNS damage. In particular, functional and genetic studies reveal that Toll-like receptor (TLR) 2 and the Nod-like receptor (NLR) P3 could be involved in MS pathogenesis, whereas TLR3, the triggering receptor expressed on myeloid cells (TREM)-2 and the C-type lectin receptors (CLRs) MBL and MASP-3 would have a putative protective role. A better understanding of these interactions will provide important insights into the aetiopathogenesis of MS and could help design potential targets for novel therapies.     

Induction of type I IFN is a physiological immune reaction to apoptotic cell-derived membrane microparticles

Membrane microparticles (MMP) released from apoptotic cells deliver signals that secure the anti-inflammatory response beyond the nearest proximity of the apoptotic cell. Plasmacytoid dendritic cells (pDC) are sentinels prepared to detect cellular processes that endanger the organism. They play a key role in the regulation of both pro- and anti-inflammatory immune responses. Based on the assumption that pDC could participate in the initiation of the anti-inflammatory response to apoptotic cells, we investigated the effects of apoptotic cell-derived MMP on human pDC. The results obtained in our experiments confirmed that MMP released from apoptotic cells trigger IFN-α secretion from human pDC. They further suggest that pDC activation results from sensing of DNA contained in MMP. MMP-DNA displays a particularly strong stimulatory activity compared with MMP-RNA and other sources of DNA. Inhibition of MMP-induced IFN-α secretion by cytochalasin D, chloroquine, and an inhibitory G-rich oligodeoxynucleotide identify TLR9 as the receptor for MMP-DNA. In marked contrast to the pDC response in autoimmune patients, in healthy subjects MMP-mediated stimulation of pDC-derived IFN-α was found to be independent of FcγRIIA (CD32A). Based on our findings, we conclude that induction of pDC-derived IFN-α by MMP is a physiological event; future investigations are necessary to elucidate whether pDC activation promotes inflammation or propagates tolerance in the context of apoptotic cell clearance.     

RP105 involved in activation of mouse macrophages via TLR2 and TLR4 signaling

RP105 is a member of the toll-like receptor family of proteins that transmits an activation signal in B cells, playing a role in regulation of B cell growth and death; in macrophages and dendritic cells, RP105 is a specific inhibitor of TLR4 signaling. RP105 is uniquely important for regulating TLR4-dependent signaling. It also proved that RP105 is closely related to TLR2 in macrophage activation by Mycobacterium tuberculosis lipoproteins. The aim of our study is to investigate the role of RP105 in mouse macrophages activation of TLR4 and TLR2 signaling by lipopolysaccharides (LPS) and Pam3CysSerLys4 (Pam3CSK4) alone or in combination, and the interaction between TLR2 and TLR4 signaling through RP105. Our results indicate that besides exhibiting negative regulation of TNF-α and IL12-p40 secretion in macrophage activated by LPS, RP105 is also involved in macrophages activation by Pam3CSK4 through TLR2 signaling and exhibited regulation to IL-10 and RANTES production by mouse peritoneal macrophage activated by Pam3CSK4. In macrophages activation by LPS and Pam3CSK4 in combination, TLR2 signaling can overcome RP105-mediated regulation of TLR4 signaling. Thus, our data demonstrate that not only TLR4 signaling, but also RP105 appears to be an essential accessory for immune responses through TLR2 signaling. The function of TLR2 and TLR4 in response to TLR ligands could be associated with each other by RP105. These results can help us understanding the unique role of RP105 in macrophages response to TLR ligands.     

Signaling via macrophage G2A enhances efferocytosis of dying neutrophils by augmentation of Rac activity

Phosphatidylserine (PS) and oxidized PS species have been identified as key ligands on apoptotic cells important for their recognition and removal (efferocytosis) by phagocytes, a requisite step for resolution of inflammation. We have recently demonstrated that lysophosphatidylserine (lyso-PS) generated and retained on neutrophils following short term activation of the NADPH oxidase in vitro and in vivo enhanced their clearance via signaling through the macrophage G-protein-coupled receptor G2A. Here, we investigated the signaling pathway downstream of G2A. Lyso-PS, either made endogenously in apoptosing neutrophils or supplied exogenously in liposomes along with lyso-PS(neg) apoptotic cells, signaled to macrophages in a G2A-dependent manner for their enhanced production of prostaglandin E2 (PGE2) via a calcium-dependent cytosolic phospholipase A2/cyclooxygenase-mediated mechanism. Subsequent signaling by PGE2 via EP2 receptors activated macrophage adenylyl cyclase and protein kinase A. These events, in turn, culminated in enhanced activity of Rac1, resulting in an increase in both the numbers of macrophages efferocytosing apoptotic cells and the numbers of cells ingested per macrophage. These data were surprising in light of previous reports demonstrating that signaling by PGE2 and adenylyl cyclase activation are associated with macrophage deactivation and inhibition of apoptotic cell uptake. Further investigation revealed that the impact of this pathway, either the enhancement or inhibition of efferocytosis, was exquisitely sensitive to concentration effects of these intermediaries. Together, these data support the hypothesis that lyso-PS presented on the surface of activated and dying neutrophils provides a tightly controlled, proresolution signal for high capacity clearance of neutrophils in acute inflammation.     

TNF-alpha inhibits macrophage clearance of apoptotic cells via cytosolic phospholipase A2 and oxidant-dependent mechanisms

Removal of apoptotic cells from inflammatory sites is an important step in the resolution of inflammation. Both murine and human macrophages stimulated with TNF-alpha or directly administered arachidonic acid showed an impaired ability to ingest apoptotic cells (efferocytosis). The inhibition was shown to be due to generation of reactive oxygen species, was blocked with a superoxide dismutase mimetic, MnTBAP, and was mimicked by direct addition of H2O2. To determine the mechanism of TNF-alpha-stimulated oxidant production, bone marrow-derived macrophages from gp91(phox)-deficient mice were examined but shown to still produce oxidants and exhibit defective apoptotic cell uptake. In contrast, a specific cytosolic phospholipase A2 inhibitor blocked the oxidant production and reversed the inhibited uptake. The suppressive effect of endogenous or exogenous oxidants on efferocytosis was mediated through activation of the GTPase, Rho. It was reversed in macrophages pretreated with C3 transferase to inactivate Rho or with an inhibitor of Rho kinase. During maturation of human monocyte-derived macrophages, only mature cells exhibited TNF-alpha-induced suppression of apoptotic cell clearance. The resistance of immature macrophages to such inhibition was shown to result not from defective generation of oxidants, but rather, from lack of response of these cells to the oxidants. Overall, the data suggest that macrophages in a TNF-alpha- and oxidant-rich inflammatory environment are less able to remove apoptotic cells and, thereby, may contribute to the local intensity of the inflammatory response.     

Role of TREM1-DAP12 in Renal Inflammation during Obstructive Nephropathy

Tubulo-interstitial damage is a common finding in the chronically diseased kidney and is characterized by ongoing inflammation and fibrosis leading to renal dysfunction and end-stage renal disease. Upon kidney injury, endogenous ligands can be released which are recognized by innate immune sensors to alarm innate immune system. A new family of innate sensors is the family of TREM (triggering receptor expressed on myeloid cell). TREM1 is an activating receptor and requires association with transmembrane adapter molecule DAP12 (DNAX-associated protein 12) for cell signaling. TREM1-DAP12 pathway has a cross-talk with intracellular signaling pathways of several Toll-like receptors (TLRs) and is able to amplify TLR signaling and thereby contributes to the magnitude of inflammation. So far, several studies have shown that TLRs play a role in obstructive nephropathy but the contribution of TREM1-DAP12 herein is unknown. Therefore, we studied TREM1 expression in human and murine progressive renal diseases and further investigated the role for TREM1-DAP12 by subjecting wild-type (WT), TREM1/3 double KO and DAP12 KO mice to murine unilateral ureter obstruction (UUO) model. In patients with hydronephrosis, TREM1 positive cells were observed in renal tissue. We showed that in kidneys from WT mice, DAP12 mRNA and TREM1 mRNA and protein levels were elevated upon UUO. Compared to WT mice, DAP12 KO mice displayed less renal MCP-1, KC and TGF-β1 levels and less influx of macrophages during progression of UUO, whereas TREM1/3 double KO mice displayed less renal MCP-1 level. Renal fibrosis was comparable in WT, TREM1/3 double KO and DAP12 KO mice. We conclude that DAP12, partly through TREM1/3, is involved in renal inflammation during progression of UUO.     

Apoptotic cell-derived metabolites in efferocytosis-mediated resolution of inflammation

The resolution of inflammation, as part of standard host defense mechanism, is the process to guarantee timely termination of inflammatory responses and eventual restoration of tissue homeostasis . It is mainly achieved via efferocytosis, during which pro-resolving macrophages clear apoptotic neutrophils at the inflammatory site. Unfortunately, impaired resolution can be the leading cause of chronic inflammatory disorders and some autoimmune diseases. Existing studies have provided relatively comprehensive understandings about the recognition and uptake of apoptotic neutrophils by macrophages during early phases of efferocytosis. However, lack of information concerns macrophage metabolism of apoptotic cell-derived metabolites after being released from phagolysosomes or the relationship between such metabolism and efferocytosis. Notwithstanding, three recent studies have revealed macrophage metabolism of cholesterol, fatty acids and arginine, as well as their respective functions in the context of inflammation-resolution. This review provides an overview of the resolution of inflammation, efferocytosis and the key players involved, followed by a focus on the metabolism of apoptotic cell-derived metabolites within efferocytes. Hypotheses of more potential apoptotic cell-derived metabolites and their possible roles in the resolution are also formulated. Understanding the effect of these metabolites further advances the concept that apoptotic cells act as active players to regulate resolution, and also suggests novel therapeutic strategies for diseases driven by defective resolution and even cancer that may be treated through enhanced efferocytosis.     

Participation of the receptor for advanced glycation end products in efferocytosis

Clearance of apoptotic cells by macrophages and other phagocytic cells, called efferocytosis, is a central process in the resolution of inflammation. Although the receptor for advanced glycation end products (RAGE) has been shown to participate in a variety of acute and chronic inflammatory processes in the lungs and other organs, a role for RAGE in efferocytosis has not been reported. In the present studies, we examined the potential involvement of RAGE in efferocytosis. Macrophages from transgenic RAGE(-/-) mice showed a decreased ability to engulf apoptotic neutrophils and thymocytes. Pretreatment of RAGE(+/+) macrophages with advanced glycation end products, which competitively bind to RAGE, or Abs against RAGE diminished phagocytosis of apoptotic cells. Overexpression of RAGE in human embryonic kidney 293 cells resulted in an increased ability to engulf apoptotic cells. Furthermore, we found that incubation with soluble RAGE enhances phagocytosis of apoptotic cells by both RAGE(+/+) and RAGE(-/-) macrophages. Direct binding of RAGE to phosphatidylserine (PS), an "eat me" signal highly expressed on apoptotic cells, was shown by using solid-phase ELISA. The ability of RAGE to bind to PS on apoptotic cells was confirmed in an adhesion assay. Decreased uptake of apoptotic neutrophils by macrophages was found under in vivo conditions in the lungs and peritoneal cavity of RAGE(-/-) mice. These results demonstrate a novel role for RAGE in which it is able to enhance efferocytosis through binding to PS on apoptotic cells.     

High mobility group protein-1 inhibits phagocytosis of apoptotic neutrophils through binding to phosphatidylserine

Phagocytosis of apoptotic cells, also called efferocytosis, is an essential feature of immune responses and critical to resolution of inflammation. Impaired efferocytosis is associated with an unfavorable outcome from inflammatory diseases, including acute lung injury and pulmonary manifestations of cystic fibrosis. High mobility group protein-1 (HMGB1), a nuclear nonhistone DNA-binding protein, has recently been found to be secreted by immune cells upon stimulation with LPS and cytokines. Plasma and tissue levels of HMGB1 are elevated for prolonged periods in chronic and acute inflammatory conditions, including sepsis, rheumatoid arthritis, acute lung injury, burns, and hemorrhage. In this study, we found that HMGB1 inhibits phagocytosis of apoptotic neutrophils by macrophages in vivo and in vitro. Phosphatidylserine (PS) is directly involved in the inhibition of phagocytosis by HMGB1, as blockade of HMGB1 by PS eliminates the effects of HMGB1 on efferocytosis. Confocal and fluorescence resonance energy transfer demonstrate that HMGB1 interacts with PS on the neutrophil surface. However, HMGB1 does not inhibit PS-independent phagocytosis of viable neutrophils. Bronchoalveolar lavage fluid from Scnn(+) mice, a murine model of cystic fibrosis lung disease which contains elevated concentrations of HMGB1, inhibits neutrophil efferocytosis. Anti-HMGB1 Abs reverse the inhibitory effect of Scnn(+) bronchoalveolar lavage on efferocytosis, showing that this effect is due to HMGB1. These findings demonstrate that HMGB1 can modulate phagocytosis of apoptotic neutrophils and suggest an alternative mechanism by which HMGB1 is involved in enhancing inflammatory responses.     

Lipopolysaccharide and double-stranded RNA up-regulate toll-like receptor 2 independently of myeloid differentiation factor 88

Toll-like receptor 2 (TLR2) is a signaling receptor for a variety of microbial products, including bacterial lipoproteins and peptidoglycan, and is central in initiating immune responses toward Gram-positive bacteria, spirochetes, and mycobacteria. The mechanisms behind regulation of TLR2 protein expression are still not well understood. By using a newly developed monoclonal antibody against mouse TLR2, we detected TLR2 protein expression on macrophages, neutrophils, and dendritic cells. Endogenous macrophage TLR2 localized mostly to the cell membrane, with particular accumulation around phagosomes containing zymosan. Treatment of macrophages with the TLR2 antibody diminished cellular response to lipoproteins and down-regulated membrane TLR2. Marked up-regulation of surface TLR2 was observed on macrophages in response to whole bacteria, lipoproteins, lipopolysaccharide, poly(I-C) (double-stranded RNA), R848, and CpG DNA, and this up-regulation appeared to be a very sensitive marker for the presence of microbial products. Up-regulation of TLR2 in response to stimuli correlated with an increased response to secondary lipoprotein exposure following a low concentration of primary lipoprotein challenge. By comparison, exposure to a larger primary challenge induced a hyporeactive state. Most interestingly, lipopolysaccharide- and double-stranded RNA-induced up-regulation of surface TLR2 in macrophages was found to be MyD88-independent, whereas the up-regulation in response to lipoproteins, R848, and CpG DNA was absent in MyD88-deficient cells. We conclude that complex mechanisms regulate expression and signaling via TLR2. Up-regulation of TLR2 in the presence of low, yet clinically relevant amounts of microbial products may be an important mechanism by which the immune system boosts its response to a beginning infection.     

IgG autoantibodies against deposited C3 inhibit macrophage-mediated apoptotic cell engulfment in systemic autoimmunity

Defective clearance of apoptotic cells has been shown in systemic lupus erythematosus (SLE) and is postulated to enhance autoimmune responses by increasing access to intracellular autoantigens. Until now, research has emphasized inherited rather than acquired impairment of apoptotic cell engulfment in the pathogenesis of SLE. In this study, we confirm previous results that efficient removal of apoptotic cells (efferocytosis) is bolstered in the presence of wild-type mouse serum, through the C3 deposition on the apoptotic cell surface. In contrast, sera from three mouse models of SLE, Mer(KD), MRL(lpr), and New Zealand Black/WF1 did not support and in fact actively inhibited apoptotic cell uptake. IgG autoantibodies were responsible for the inhibition, through the blockade of C3 recognition by macrophages. Consistent with this, IgG removal reversed the inhibitory activity within autoimmune serum, and purified autoimmune IgG blocked both the detection of C3 on apoptotic cells and C3-dependent efferocytosis. Sera from SLE patients demonstrated elevated anti-C3b IgG that blocked detection of C3 on apoptotic cells, activity that was not found in healthy controls or patients with rheumatoid arthritis, nor in mice prior to the onset of autoimmunity. We propose that the suppression of apoptotic cell disposal by Abs against deposited C3 may contribute to increasing severity and/or exacerbations in SLE.     

Extracellular histones inhibit efferocytosis

The uptake and clearance of apoptotic cells by macrophages and other phagocytic cells, a process called efferocytosis, is a major component in the resolution of inflammation. Increased concentrations of extracellular histones are found during acute inflammatory states and appear to contribute to organ system dysfunction and mortality. In these studies, we examined the potential role of histones in modulating efferocytosis. We found that phagocytosis of apoptotic neutrophils or thymocytes by macrophages was significantly diminished in the presence of histones H3 or H4, but not histone H1. Histone H3 demonstrated direct binding to macrophages, an effect that was diminished by preincubation of macrophages with the opsonins growth arrest–specific gene 6 (Gas6) and milk fat globule–epidermal growth factor (EGF) 8 (MFG-E8). Incubation of histone H3 with soluble α_vβ₅ integrin and Mer, but not with α_vβ₃, diminished its binding to macrophages. Phagocytosis of apoptotic cells by alveolar macrophages in vivo was diminished in the presence of histone H3. Incubation of histone H3 with activated protein C, a treatment that degrades histones, abrogated its inhibitory effects on efferocytosis under both in vitro and in vivo conditions. The present studies demonstrate that histones have inhibitory effects on efferocytosis, suggesting a new mechanism by which extracellular histones contribute to acute inflammatory processes and tissue injury.     

The receptor for urokinase regulates TLR2 mediated inflammatory responses in neutrophils

The urokinase-type plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol (GPI) anchored membrane protein, regulates urokinase (uPA) protease activity, chemotaxis, cell-cell interactions, and phagocytosis of apoptotic cells. uPAR expression is increased in cytokine or bacteria activated cell populations, including macrophages and monocytes. However, it is unclear if uPAR has direct involvement in the response of inflammatory cells, such as neutrophils and macrophages, to Toll like receptor (TLR) stimulation. In this study, we found that uPAR is required for optimal neutrophil activation after TLR2, but not TLR4 stimulation. We found that the expression of TNF-α and IL-6 induced by TLR2 engagement in uPAR-/- neutrophils was less than that in uPAR+/+ (WT) neutrophils. Pretreatment of neutrophils with PI-PLC, which cleaves GPI moieties, significantly decreased TLR2 induced expression of TNF-α in WT neutrophils, but demonstrated only marginal effects on TNF-α expression in PAM treated uPAR-/- neutrophils. IκB-α degradation and NF-κB activation were not different in uPAR-/- or WT neutrophils after TLR2 stimulation. However, uPAR is required for optimal p38 MAPK activation after TLR2 engagement. Consistent with the in vitro findings that uPAR modulates TLR2 engagement induced neutrophil activation, we found that pulmonary and systemic inflammation induced by TLR2, but not TLR4 stimulation is reduced in uPAR-/- mice compared to WT counterparts. Therefore, our data suggest that neutrophil associated uPAR could be a potential target for treating acute inflammation, sepsis, and organ injury related to severe bacterial and other microbial infections in which TLR2 engagement plays a major role.