Petra Heyder

Induction of inflammatory and immune responses by HMGB1–nucleosome complexes: implications for the pathogenesis of SLE

Autoantibodies against double-stranded DNA (dsDNA) and nucleosomes represent a hallmark of systemic lupus erythematosus (SLE). However, the mechanisms involved in breaking the immunological tolerance against these poorly immunogenic nuclear components are not fully understood. Impaired phagocytosis of apoptotic cells with consecutive release of nuclear antigens may contribute to the immune pathogenesis. The architectural chromosomal protein and proinflammatory mediator high mobility group box protein 1 (HMGB1) is tightly attached to the chromatin of apoptotic cells. We demonstrate that HMGB1 remains bound to nucleosomes released from late apoptotic cells in vitro. HMGB1–nucleosome complexes were also detected in plasma from SLE patients. HMGB1-containing nucleosomes from apoptotic cells induced secretion of interleukin (IL) 1β, IL-6, IL-10, and tumor necrosis factor (TNF) α and expression of costimulatory molecules in macrophages and dendritic cells (DC), respectively. Neither HMGB1-free nucleosomes from viable cells nor nucleosomes from apoptotic cells lacking HMGB1 induced cytokine production or DC activation. HMGB1-containing nucleosomes from apoptotic cells induced anti-dsDNA and antihistone IgG responses in a Toll-like receptor (TLR) 2–dependent manner, whereas nucleosomes from living cells did not. In conclusion, HMGB1–nucleosome complexes activate antigen presenting cells and, thereby, may crucially contribute to the pathogenesis of SLE via breaking the immunological tolerance against nucleosomes/dsDNA.

Complement binding is an early feature of necrotic and a rather late event during apoptotic cell death

The phagocytosis of dying cells is an integral feature of apoptosis and necrosis. There are many receptors involved in recognition of dying cells, however, the molecular mechanisms of the scavenging process remain elusive. The activation by necrotic cells of complement is well established, however, the importance of complement in the scavenging process of apoptotic cells was just recently described. Here we report that the complement components C3 and C4 immediately bound to necrotic cells. The binding of complement was much higher for lymphocytes compared to granulocytes. In case of apoptotic cell death complement binding was a rather late event, which in lymphocytes was preceded by secondary necrosis. Taken together complement binding is an immediate early feature of necrosis and a rather late event during apoptotic cell death. We conclude that complement may serve as an opsonin for fragments of apoptotic cells that have escaped regular scavenging mechanisms. Cell Death and Differentiation (2001) 8, 327–334

Autoantigens are translocated into small apoptotic bodies during early stages of apoptosis.

A dysregulation of apoptosis or an ineffective clearance of apoptotic material is suspected to be involved in the pathogenesis of systemic lupus erythematodes. Subcellular fragments such as apoptotic bodies (ABs) have been recognized as modulators of intercellular communication and immune function. In this context, we have been interested whether nuclear and cytoplasmic antigens are relocated into ABs. In the present study, we characterized ABs isolated from apoptozing lymphoblasts. We found an accumulation of the linker-histone (histone 1) as well as the core-histones (histone 2A, histone 2B, histone 3, histone 4) in ABs. Further, they contained DNA, RNA and the ribonuclear protein La/SSB. Proteins such as cytochrome c, HSP 70, prohibitin, p53, nuclear matrix antigen or lamin B were excluded from ABs. The content of ABs differed from that observed in membrane microparticles isolated from viable cells. Formation of ABs occurred early during apoptosis. It was observed before DNA-degradation or phosphatidylserine exposure was detected. ABs were engulfed by monocyte-derived phagocytes. These findings suggest that immunogenic molecules are actively translocated into ABs followed by a rapid engulfment of the latter by environmental phagocytes. In autoimmune diseases, a defect in the clearance of ABs or AB formation may contribute to the development of autoimmunity.

Decrease of sialic acid residues as an eat-me signal on the surface of apoptotic lymphocytes

The silent clearance of apoptotic cells is essential for cellular homeostasis in multicellular organisms, and several mediators of apoptotic cell recognition have been identified. However, the distinct mechanisms involved are not fully deciphered yet. We analyzed alterations of the glycocalyx on the surfaces of apoptotic cells and its impact for engulfment. After apoptosis induction of lymphocytes, a decrease of α2,6-terminal sialic acids and sialic acids in α2,3-linkage with galactose was observed. Similar changes were to be found on the surface of apoptotic membrane blebs released during early stages of apoptosis, whereas later released blebs showed no impaired, but rather an increased, exposure of sialic acids. We detected an exposure of fucose residues on the surface of apoptotic-cell-derived membrane blebs. Cleavage by neuraminidase of sialic acids, as well as lectin binding to sialic acids on the surfaces, enhanced the engulfment of apoptotic cells and blebs. Interestingly, even viable lymphoblasts were engulfed in an autologous cell system after neuraminidase treatment. Similarly, the engulfment of resting apoptotic lymphocytes was augmented after neuraminidase treatment. However, the engulfment of resting viable lymphocytes was not significantly enhanced after neuraminidase treatment. Our findings support the importance of the glycocalyx, notably the terminal sialic acids, in the regulation of apoptotic cell clearance. Thus, depending on cell type and activation status, changes in surface glycosylation can either directly mediate cellular engulfment or enhance phagocytosis by cooperation with further engulfment signals.

Exposure of anionic phospholipids serves as anti-inflammatory and immunosuppressive signal--implications for antiphospholipid syndrome and systemic lupus erythematosus.

In contrast to necrotic cells, the clearance of apoptotic ones usually is an anti-inflammatory process which elicits only a marginal immune response. During apoptosis phosphatidylserine (PS) is exposed on the outer leaflet of the cytoplasmic membrane and serves as target for the PS receptor of phagocytes. The latter is responsible for anti-inflammatory signalling and the induction of TGFbeta. We were interested whether the immunogenicity of apoptotic cells can be increased by masking PS. We observed that treatment of xenogeneic apoptotic cells with annexin V (AxV) significantly increased the humoral immune response against surface epitopes of these cells. Furthermore, AxV-coated irradiated tumour cells were able to elicit a long lasting tumour specific cytotoxic T lymphocyte response. AxV efficiently blocked the uptake of irradiated cells by macrophages but not by dendritic cells. Furthermore, AxV skewed the phagocytosis of irradiated cells towards inflammation. Investigation of patients with autoimmune diseases further supported the role of anionic surface phospholipids for anti-inflammatory clearance of apoptotic cells. Impaired clearance and opsonisation with anti-phospholipid-antibodies are discussed to be responsible for the development of systemic lupus erythematosus and anti-phospholipid-syndrome, respectively. Presentation of cryptic epitopes from late apoptotic cells in a proinflammatory context may challenge T cell tolerance. In addition, accumulation of uncleared apoptotic debris in the germinal centres of lymph nodes may result in the survival of autoreactive B cells.

Clinical manifestations but not cytokine profiles differentiate adult-onset Still's disease and sepsis.

Objective. To analyze clinical manifestations, serum ferritin, and serum cytokine levels in patients with adult-onset Still’s disease (AOSD) or bacterial sepsis and to evaluate their potential use for differential diagnosis. Methods. Twenty-two consecutive patients with the first flare of AOSD and 6 patients with an established diagnosis of AOSD under immunosuppressive therapy were compared with 14 patients with bacterial sepsis. Clinical manifestations were scored in a Pouchot AOSD activity score including elevated serum ferritin levels to obtain a modified Pouchot score. Serum cytokine profiles were analyzed from each patient. Results. The scores of clinical manifestations using a modified Pouchot activity score were significantly higher in patients with active untreated AOSD (mean 5.60 ± 1.93) compared with patients with chronic AOSD (mean 1.16 ± 0.98; p < 0.001) and patients with sepsis (mean 2.38 ± 1.19; p < 0.001). A modified Pouchot score ≥ 4 shows a sensitivity of 92% and a specificity of 93% for active AOSD. Serum cytokine levels of interleukin 1ß (IL-1ß), IL-6, IL-8, IL-10, IL-12, IL-18, interferon-γ, tumor necrosis factor-α, and calprotectin were elevated in acute AOSD and sepsis. Significant differences were detected only in patients with sepsis who had higher levels of IL-6 and IL-8. The overlap of the 2 groups limits the use of cytokines for differential diagnosis in individual patients. Conclusion. A modified Pouchot AOSD activity score including elevated serum ferritin levels was more useful to confirm the diagnosis of AOSD compared to patients with sepsis. Elevated serum cytokines correlate with inflammation but are of limited use to differentiate between active AOSD and bacterial sepsis.

Early detection of apoptosis by staining of acid-treated apoptotic cells with FITC-labeled lectin from Narcissus pseudonarcissus.

Exposure of anionic phospholipids and modified carbohydrates are main parts of the apoptotic death program. Cells undergoing apoptosis can be identified by various methods, detecting surface changes or modifications of their organelles, respectively. We describe a method for the detection of early apoptosis by staining of cells with fluorescein isothiocyanate (FITC)–labeled lectin from Narcissus pseudonarcissus (NPn).

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.

UV irradiation inhibits ABC transporters via generation of ADP-ribose by concerted action of poly(ADP-ribose) polymerase-1 and glycohydrolase.

AbstractATP-binding cassette (ABC) transporters are involved in the transport of multiple substrates across cellular membranes, including metabolites, proteins, and drugs. Employing a functional fluorochrome export assay, we found that UVB irradiation strongly inhibits the activity of ABC transporters. Specific inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) restored the function of ABC transporters in UVB-irradiated cells, and PARP-1-deficient cells did not undergo UVB-induced membrane transport inhibition. These data suggest that PARP-1 activation is necessary for ABC transporter functional downregulation. The hydrolysis of poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase (PARG) was also required, since specific PARG inhibitors, which limit the production of ADP-ribose molecules, restored the function of ABC transporters. Furthermore, ADP-ribose molecules potently inhibited the activity of the ABC transporter P-glycoprotein. Hence, poly(ADP-ribose) metabolism appears to play a novel role in the regulation of ABC transporters.

During apoptosis HMGB1 is translocated into apoptotic cell-derived membraneous vesicles

High mobility group box protein B1 (HMGB1), a nuclear protein reportedly involved in the structural organisation of DNA, is released from necrotic cells or upon cellular activation. After its release into the extracellular space, HMGB1 serves as a mediator of inflammation. In contrast to necrotic cells, apoptotic ones usually do not release HMGB1. Formation and release of membraneous vesicles is a well-known feature of apoptotic cell death. Only recently, subcellular membrane vesicles, such as those released during apoptotic cell death have been identified as immune regulators and as mediators of cell to cell communication. We and others have previously detected nuclear antigens within apoptosis-released membranous vesicles and HMGB1 together with nuclear antigens has been discussed to be a key player in etiology and pathogenesis of autoimmune diseases. On this background, we analysed whether HMGB1 is included in the membraneous vesicles generated by apoptosing cells. Employing immune blots we observed abundand amounts of HMGB1 in the fraction of the small membraneous particles isolated from cell culture supernatants and conclude that HMGB1 is translocated into vesicles generated during apoptosis.