Apoptotic-cell-derived membrane microparticles and IFN-α induce an inflammatory immune response.

A dysregulation in the clearance of apoptotic material is considered a major pathogenetic factor for the emergence of autoimmune diseases. Apoptotic-cell-derived membrane microparticles (AdMPs), which are released from the cell surface during apoptosis, have been implicated in the pathogenesis of autoimmunity. Also of importance are cytokines, such as interferon-α (IFN-α), which is known to be a major player in patients with systemic lupus erythematosus (SLE). This study investigates the combined effect of AdMPs and IFN-α on professional phagocytes. In the presence of IFN-α, phagocytosis of AdMPs by human monocytes was significantly increased in a dose-dependent manner. The combination of AdMPs and raised IFN-α concentrations resulted in an increase in the secretion of pro-inflammatory cytokines and an upregulation of surface molecule expression involved in antigen uptake. In addition, macrophage polarisation was shifted towards a more inflammatory type of cell. The synergism between IFN-α and AdMPs seemed to be mediated by an upregulation of phosphorylated STAT1. Our results indicate that IFN-α, together with AdMPs, amplify the initiation and maintenance of inflammation. This mechanism might especially play a crucial role in disorders with a defective clearance of apoptotic material.

During apoptosis HMGB1 is translocated into apoptotic cell-derived membranous 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 membranous 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 membranous 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.

Apoptotic-cell-derived membrane vesicles induce an alternative maturation of human dendritic cells which is disturbed in SLE.

The clearance of apoptotic cells occurs in a non-inflammatory context. Defects in this clearance process have been linked to the emergence of human autoimmune diseases like systemic lupus erythematosus (SLE). A characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surfaces. Those vesicles have recently been recognized as mediators of intercellular communication or as adjuvant in the pathogenesis of autoimmune diseases. We analyzed the interactions between these apoptotic cell-derived membrane vesicles and professional antigen presenting cells. These vesicles were engulfed by monocyte-derived dendritic cells (mDC) and stimulated their maturation towards a phenotype comprising an upregulation of CD80, CD83, CD86, and a remarkable downregulation of MHC class II molecules. We observed only a minor release of proinflammatory cytokines from these mDC when compared to LPS stimulation. mDC stimulated by apoptotic vesicles did not cause significant T-cell expansion. Interestingly, when compared to normal healthy donors SLE patients-derived dendritic cells showed a significantly different phenotype lacking the downregulation of MHC class II, which correlated to disease activity.

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.

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.

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.

Cells induced to undergo apo in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone stimulate Mo derived phagocytes to secrete proinflammatory cytokines.

In contrast to nec, the apo is not accompanied by local inflammation. The immunosuppressive effects of apo cells have been repeatedly reported and a dysregulation of apo is discussed to play a major role in the pathogenesis of autoimmune disorders. The intracellular executioners of apo are the cysteine-aspartic acid proteases, also known as caspases that cleave a variety of intracellular substrates and mediate the morphological changes observed during apo. The association of autoimmune diseases with defects in caspase function indicates the necessity for functional integrity of caspases in the apo cell death machinery. Here, we describe that cells undergoing apo in presence of the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone stimulated MPhi to secrete proinflammatory cytokines. These findings indicate that caspase signalling is of central importance for silent and non- or anti-inflammatory cell death.

TLR9-activating DNA up-regulates ZAP70 via sustained PKB induction in IgM+ B cells.

In the past, ZAP70 was considered a T cell-specific kinase, and its aberrant expression in B-CLL cells was interpreted as a sign of malignant transformation and dedifferentiation. It was only recently that ZAP70 was detected in normal human B cells. In this study, we show that TLR9-activated B cells resemble B-cell chronic lymphocytic leukemia cells with regard to CD5, CD23, CD25, and heat shock protein 90 expression. Furthermore, stimulatory CpG and GpC DNA oligonucleotides target CD27(+)IgM(+) and CD27(-)IgM(+) B cells (but not IgM(-) B cells) and enhance ZAP70 expression predominantly in the IgM(+)CD27(+) B cell subset. ZAP70 is induced via activation of TLR-7 or -9 in a MyD88-dependent manner, depends on protein kinase B (PKB)/mammalian target of rapamycin signaling and is rapamycin sensitive. Furthermore, ZAP70 expression levels correlate with induction of cyclin A2, prolonged B cell proliferation, and sustained induction of PKB. These events are not observed upon CD40 ligation. However, this deficit can be overcome by the expression of constitutively active PKB, given that CD40 ligation of PKB-transgenic B cells induces B cell proliferation and ZAP70 expression. These results highlight a major difference between CD40- and TLR-7/9-mediated B cell activation and suggest that ZAP70 expression levels in B cells give an estimate of the proliferative potential and the associated PKB availability.

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) 1beta, IL-6, IL-10, and tumor necrosis factor (TNF) alpha 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.

Hypothesis: human serum-borne albumin bound lipids promote cellular survival after apoptosis induction by a variety of stimuli.

A tight control of proliferation and cell death is required to maintain homeostasis in multicellular organisms. Several specific pro- and anti-apoptotic regulators and pathways have been deciphered being responsible for these complex tasks. Here we describe a human serum-borne activity promoting cellular fitness and inhibiting apoptosis after a plethora of different cell death stimuli. The factor(s) do not inhibit a specific death pathway, instead it/they can be considered as general pro-survival factor(s) for cultured cells. The activity is heat stable (30 min, 96 degrees C), co-migrates with albumin in size exclusion chromatography, and is sensitive to chemical delipidation. A similar activity is observed in native, non-delipidated preparations of human albumin, while delipidated albumin is not effective. These properties point to heat stable factors that exert anti-apoptotic activities, most likely albumin bound bioactive lipids. The activity prevented Akt dephosphorylation and degradation, after apoptosis induction by staurosporine and the production of reactive oxygen species after UV-B irradiation. In conclusion human serum-enriched bioactive lipids promote survival of cultured cells overriding the pro-apoptotic effects of a variety of apoptosis inducing agents.

The CFSE distribution assay is a powerful technique for the analysis of radiation-induced cell death and survival on a single-cell level.

BACKGROUND AND PURPOSE: To analyze radiation sensitivity of cells and to monitor cellular responses to irradiation, sensitive test systems for cell death and proliferation on a single-cell level are required. Traditionally, cellular radiation survival is measured using the clonogenic assay as the gold standard. Here it is reported, that labeling of cells with 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester (CFDASE) can be used as a highly sensitive assay to determine cellular response toward irradiation on a single-cell level. MATERIAL AND METHODS: The human malignant cell lines U937 (myelomonocytic, nonadherent), SW48 and SW480 (colorectal, adherent) were labeled with CFDASE, irradiated with either UVB (0-540 mJ/cm(2)), or X-rays (0-16 Gy). Cell death and proliferation were monitored by cytofluorometry and compared to the clonogenic assay for adherent SW48 and SW480 cells. RESULTS: Dividing nonadherent U937 cells displayed a shift in carboxyfluorescein (CF) fluorescence in parallel with an increased cell count indicating cell proliferation. By comparison, UVB-irradiated U937 cells did not show a shift in CF fluorescence and an increase in cell count indicating cell-cycle arrest. In a mixed cell culture, only the nonirradiated cells divided and concomitantly reduced their fluorescence. Calculating the number of cell divisions it was observed that the nonirradiated cells underwent approximately six cell divisions within 7 days, whereas the irradiated cells divided only once on average. The adherent SW480 colorectal cells showed a more pronounced cell-cycle arrest after irradiation with 240 mJ/cm(2) UVB as compared to cells treated with X-ray up to 16 Gy. Furthermore, the CFSE assay also discriminated colorectal cell lines of different intrinsic radiosensitivities and yielded results comparable to the standard clonogenic assay. CONCLUSION: Analysis of CF distribution can be employed as a powerful add-on to the clonogenic assay to simultaneously monitor cellular responses toward irradiation on a single-cell level. It constitutes an add-on to the clonogenic assay, especially for nonadherent cells.

Loss of GM1 surface expression precedes annexin V-phycoerythrin binding of neutrophils undergoing spontaneous apoptosis during in vitro aging.

BACKGROUND: Apoptosis of neutrophil granulocytes is an important determinant of the resolution of inflammation. Apoptotic neutrophils undergo specific alterations in their receptor profiles. These alterations are likely to contribute to the characteristic functional silencing of the dying cells. METHODS: By flow cytometry and fluorescence microscopy, we analyzed the ganglioside GM1, a lipid raft marker, with respect to its surface expression on neutrophil and eosinophil granulocytes. Apoptosis was monitored by morphological changes and by the binding of annexin V-phycoerythrin (AxV-PE). RESULTS: GM1, which was stained by the cholera toxin subunit B, was found only on neutrophil granulocytes; eosinophil granulocytes did not bind cholera toxin subunit B. GM1 was lost from the surfaces of neutrophils before AxV-PE binding (early apoptosis). Surprisingly, GM1 reappeared during the late stages of apoptosis, although without functional consequences. GM1 was found on the cell surface and in intracellular membranes, whereas CD16 was found only at the cell surface. CONCLUSIONS: Loss of surface GM1 is a new marker for the detection of the aging of neutrophils. Its loss precedes the binding of AxV-PE of neutrophils.

Cooperation between C1q and DNase I in the clearance of necrotic cell-derived chromatin.

OBJECTIVE: The efficient uptake of dying cells by phagocytes is essential to the avoidance of chronic inflammation. Some human autoimmune responses are thought to be driven by autoantigens from apoptotic or necrotic cells. We analyzed the role of C1q and DNase I in the disposal of necrotic cell-derived chromatin because deficiencies in these serum factors predispose to the development of systemic autoimmune disorders, such as systemic lupus erythematosus. METHODS: Human necrotic peripheral blood lymphocytes were incubated in cell culture medium supplemented with various sera or serum components. Chromatin degradation was monitored by measuring the residual DNA content by flow cytometry. The uptake of necrotic cell-derived nuclei was analyzed by in vitro phagocytosis assays. RESULTS: Reconstitution of C1q-depleted serum with C1q strongly increased its ability to degrade necrotic cell-derived chromatin. Although C1q itself displayed no DNase activity, it strongly augmented the activity of serum DNase I. Whereas an excess of recombinant DNase I degraded chromatin in the absence of C1q, efficient uptake of the predigested material by monocyte-derived phagocytes required the presence of C1q. These data show that C1q and DNase I cooperate in the degradation of chromatin from necrotic cells. Furthermore, C1q was found to be necessary for effective uptake of degraded chromatin by monocyte-derived phagocytes. CONCLUSION: C1q or DNase I deficiencies may precipitate autoimmunity in humans by a mechanism similar to that of other molecules that are involved in the safe, efficient, and rapid disposal of dying cells.

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

BACKGROUND: 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). METHODS: Apoptosis in cells or in cell lines was induced by various stimuli. To detect apoptosis the cells were stained with FITC-labeled lectin of NPn. After a short-term acid treatment they were analyzed by flow cytometry. RESULTS: The instability of the cytoplasmic membrane against acid and the binding of NPn were very early features of apoptotic cell death. The NPn lectin staining procedure detected apoptosis with high sensitivity. The staining was stable for at least 12 h. CONCLUSIONS: The method described in this study is suitable for the detection of the very early phases of apoptosis. The NPn lectin staining after short-term acid treatment can, therefore, be added to the list of reliable tools for the research of cell death.

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.