Apoptotic cells were shown to induce dendritic cell immune tolerance. We applied a proteomic approach to identify molecules that are secreted from apoptotic monocytes, and thus may mediate engulfment and immune suppression. Supernatants of monocytes undergoing apoptosis were collected and compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and differentially expressed proteins were identified using tandem mass spectrometry. Thrombospondin-1 (TSP-1) and its cleaved 26-kDa heparin-binding domain (HBD) were identified. We show that TSP-1 is expressed upon induction of monocyte apoptosis in a caspase-dependent pattern and the HBD is cleaved by chymotrypsinlike serine protease. We further show that CD29, CD36, CD47, CD51, and CD91 simultaneously participate in engulfment induction and generation of an immature dendritic cell (iDC) tolerogenic and phagocytic state. We conclude that apoptotic cell TSP-1, and notably its HBD, creates a signalosome in iDCs to improve engulfment and to tolerate engulfed material prior to the interaction with apoptotic cells. IntroductionIn recent years, it has become apparent that upon induction of apoptosis, apoptotic cells play an active role in their own engulfment by signaling professional phagocytes and/or antigenpresenting cells, without triggering an inflammatory or autoimmune response. [1][2][3][4][5] This process seems to play an important role in homeostasis, resolution of inflammation, and peripheral tolerance induction. 4,[6][7][8] Apoptotic cells have been shown to signal the innate immune system in a variety of ways. "Eat me" signals on apoptotic cells serve as markers for phagocytes to specifically recognize these cells and subsequently ingest them. Such signals can appear on apoptotic cell membranes. Direct signals include alteration in cell surface phospholipid composition, 9 changes in cell surface glycoprotein expression, distinct alterations in cell surface charge, 10,11 or expression of specific molecules. 12 Alternatively, certain serum or phagocyte-derived proteins can opsonize an apoptotic cell surface and signal phagocytes to engulf the opsonized cells. 4,[13][14][15][16][17] Viable cells actively express "do not eat me" signals by restriction of phosphatydilserine to the inner leaflet of their membrane, or "stay away" signals using CD31 expression. 18 Recently, attention has been given not only to apoptotic cell membrane changes and phagocyte receptors, but also to the release of a membrane-derived phospholipid, lysophosphatidylcholine, which acts as a "find me" signal that is important for phagocytic cell recruitment. 19 Most of these mechanisms suggest efficient identification and clearance of cells undergoing apoptosis, with noninflammatory and nonautoimmune consequences.We decided to further explore whether apoptotic cells can actively express and secrete molecules that have a physiological significance for their own engulfment and for the environmental immune suppression. We examined whether apoptosis-induced immune suppress...

IsraelIn recent years, it has become apparent that the removal of apoptotic cells by macrophages and DC is not only noninflammatory, but also immune-inhibitory, in most although not all circumstances. Complement may be involved in the uptake of apoptotic cells via direct binding of bridging factors in some physiological circumstances, by opsonization and engagement of the complement receptors. In the current study, we use a complementdependent system of apoptotic cell clearance by human-derived macrophages and DC. Using a luciferase reporter gene and measuring immune response to non-opsonic zymosan, we show that iC3b-apoptotic cells induce NF-jB inhibition in response to zymosan and LPS at the nuclear translocation, transcriptional and post-transcriptional levels, leading to profound inhibition of proinflammatory cytokines. In addition, interaction with iC3b-opsonized apoptotic cells is characterized by macrophage secretion of IL-10 and lack of TGF-b secretion.In conclusion, in cells with iC3b receptors, opsonized apoptotic cells mediate a distinct antiinflammatory response and transcriptional NF-jB-dependent blockage.Key words: Apoptosis . Complement . iC3b . NF-kB IntroductionCells undergoing apoptosis in the human body express cell surface changes that allow recognition by professional phagocytes and/or neighboring cells. Removal of these cells occurs rapidly and without induction of a proinflammatory milieu [1]. In recent years, it has become apparent that the removal of apoptotic cells by macrophages and DC is not only noninflammatory but also immune-inhibitory [2][3][4][5][6][7][8], in most although not all circumstances. Fadok et al. [2] showed that efferocytosis (clearance of apoptotic cells, a terminology suggested by the Henson group) inhibited the production of proinflammatory cytokines such as IL-8 and IL-1b, and induced the secretion of TGF-b, platelet-activating factor, and prostaglandin E2. They further showed and suggested that these factors inhibited a proinflammatory response to LPS and zymosan, by autocrine or paracrine mechanisms, via the secreted factors. Later, Huynh et al. [4] showed that the resolution of acute inflammation is dependent on phosphatidylserine expressed by apoptotic cells, and on TGF-b, secreted most probably by macrophages following engulfment of apoptotic cells expressing phosphatidylserine. further showed that through TGF-b, apoptotic cells simultaneously induce an anti-inflammatory milieu and suppress proinflammatory eicosanoid and NO synthesis in murine macrophages. Hence, the proposed model for à These authors contributed equally to this work. inhibition of a proinflammatory response to LPS and zymosan, as well as the resolution of acute inflammation, is based on ligation of phosphatidylserine expressed on apoptotic cells to the presumed phosphatidylserine receptor, and possibly other receptors. This ligation is expected to result in immediate preformed TGF-b secretion from macrophages, followed by de novo synthesis of TGF-b. Additional mechanisms of inflammatory res...

Programmed cell death (PCD) is a fundamental mechanism in tissue and cell homeostasis. It was long suggested that apoptosis regulates the cell number in diverse cell populations; however no clear mechanism was shown. Neutrophils are the short-lived, first-line defense of innate immunity, with an estimated t = 1/2 of 8 hours and a high turnover rate. Here we first show that spontaneous neutrophil constitutive PCD is regulated by cell concentrations. Using a proteomic approach, we identified the S100 A8/9 complex, which constitutes roughly 40% of cytosolic protein in neutrophils, as mediating this effect. We further demonstrate that it regulates cell survival via a signaling mechanism involving MEK-ERK via TLR4 and CD11B/CD18. This mechanism is suggested to have a fine-tuning role in regulating the neutrophil number in bone marrow, peripheral blood, and inflammatory sites.

SummaryDendritic cell (DC) maturation may accelerate autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, and may contribute to accelerated atherosclerosis seen in these patients. The immune system responds to both exogenous and endogenous 'dangerous' signals that can induce dendritic cell maturation. We have found that autologous plasma contains danger signals that induce up-regulation of major histocompatibility complex (MHC) class II and co-stimulatory molecules in immature DCs (iDCs). The objective of this study was to determine whether low-density lipoprotein (LDL) and/or oxidized LDL (oxLDL) constitute danger signals, and to assess the effect of exposure to LDL and oxLDL following monocyte differentiation into iDCs in lipoprotein-deficient serum (LPDS). IDCs were generated in the presence of autologous plasma or LPDS. Expression of maturation and migration molecules was evaluated using flow cytometry, and morphology was assessed by light microscopy. Pro-or anti-apoptotic effect was determined using annexin V and propidium iodide binding. Phagocytosis of apoptotic cells was evaluated using autologous plasma or LPDS. LDL and oxLDL were clearly able to slightly up-regulate levels of HLA-DR and co-stimulatory molecule CD86. High oxLDL concentrations (50-100 mg/ml) were associated with expression of additional maturation molecules. Moreover, iDCs that were prepared in LPDS showed partial maturation following exposure to LDL and oxLDL, and improved tolerogenic apoptotic cell uptake. This study suggests that oxLDL, and to some extent LDL, are at least partly responsible for the iDC 'danger' response induced by autologous plasma.

One hallmark of programmed cell death (PCD) is redistribution of phosphatidylserine (PS) to the plasma membrane's outer leaflet. Annexin V is widely used in cell death research due to its calcium-dependent ability to bind phosphatidylserine, thus marking apoptotic cells. However, calcium is invariably used at high concentrations in annexin V staining, at doses that can induce cell death. We used flow cytometric annexin V staining, together with propidium iodide and TMRM for determination of dissipation of mitochondrial potential, with a variety of calcium concentrations, cell media, and incubation times, to identify a possible bias in PCD determination of human primary leukocytes. Here we show that measurements of PCD in human monocytes, polymorphonuclear cells, and monocyte-derived dendritic cells using annexin V may be dramatically affected by calcium concentration, time of incubation on ice, and media choice. We propose a method that enables accurate and unbiased annexin V staining, without affecting results.

To reach the lymphatics, migrating dendritic cells (DCs) need to interact with the extracellular matrix (ECM). Heparanase, a mammalian endo-β-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and ECM. The role of heparanase in the physiology of bone marrow-derived DCs was studied in mutant heparanase knock-out (Hpse-KO) mice. Immature DCs from Hpse-KO mice exhibited a more mature phenotype; however their transmigration was significantly delayed, but not completely abolished, most probably due to the observed upregulation of MMP-14 and CCR7. Despite their mature phenotype, uptake of beads was comparable and uptake of apoptotic cells was more efficient in DCs from Hpse-KO mice. Heparanase is an important enzyme for DC transmigration. Together with CCR7 and its ligands, and probably MMP-14, heparanase controls DC trafficking.

Human monocyte-derived dendritic cells (mdDCs) are versatile cells that are used widely for research and experimental therapies. Although different culture conditions can affect their characteristics, there are no known subpopulations. Since monocytes differentiate into dendritic cells (DCs) in a variety of tissues and contexts, we asked whether they can give rise to different subpopulations. In this work we set out to characterize two human mdDC subpopulations that we identified and termed small (DC-S) and large (DC-L). Morphologically, DC-L are larger, more granular and have a more complex cell membrane. Phenotypically, DC-L show higher expression of a wide panel of surface molecules and stronger responses to maturation stimuli. Transcriptomic analysis confirmed their separate identities and findings were consistent with the phenotypes observed. Although they show similar apoptotic cell uptake, DC-L have different capabilities for phagocytosis, demonstrate better antigen processing, and have significantly better necrotic cell uptake. These subpopulations also have different patterns of cell death, with DC-L presenting an inflammatory, “dangerous” phenotype while DC-S mostly downregulate their surface markers upon cell death. Apoptotic cells induce an immune-suppressed phenotype, which becomes more pronounced among DC-L, especially after the addition of lipopolysaccharide. We propose that these two subpopulations correspond to inflammatory (DC-L) and steady-state (DC-S) DC classes that have been previously described in mice and humans.

Orbital (slow flow) cavernous venous hemangiomas (OCVH) are the most common benign orbital tumors in adults. The c-KIT is a tyrosine kinase receptor, which is expressed on several types of cells, is thought to play a key role in tumor pathogenesis. The purpose of this study was to evaluate the presence of the receptor c-KIT in OCVH. Our retrospective study examined 16 orbital cavernous venous hemangiomas from 16 cases operated on between 2006–2016 at Emek Medical Center. The mean tumor size was 18.4 mm. Symptoms appeared between 6 months and 22 years before operation. All specimens were analyzed for the c-KIT receptor through immunohistochemistry. The c-KIT was expressed by the endothelium in all 16 preparates. Staining was strong in two cases, moderate in six, and weak in eight cases, with no statistically significant correlation between staining and tumor size (p = 0.69) or the symptom duration (p = 0.15). We conclude that c-KIT may play an important role in the pathogenesis of OCVH. This pilot study is significant in that tumor-targeted therapy such as Imatinib Mesylate and Sunitinib may have a role in treating surgically complicated cases located in the orbital apex. A large multicenter collaborative study is necessary to examine the role of c-KIT in OCVH.