Martin Schiller

The role of defective clearance of apoptotic cells in systemic autoimmunity

The inefficient clearance of dying cells can result in the accumulation of apoptotic cell remnants. This occurrence is considered an intrinsic defect that can cause the permanent presence of cellular debris responsible for the initiation of systemic autoimmunity in diseases such as systemic lupus erythematosus (SLE). If postapoptotic debris accumulates in germinal centers, activates complement and functions as a survival signal for B cells that have become autoreactive by somatic hypermutation, autoimmunity could arise (etiology). The accumulation of postapoptotic remnants and fragments derived from secondary necrotic cells in the presence of autoantibodies against apoptotic cells or adaptor molecules obliges their pathological elimination and maintains autoinflammation. The autoimmunity that occurs in patients with SLE involves complex antigens that contain nucleic acids, which can function as virus mimetics. Complexes of autoantibodies, proteins and nucleic acids are likely to be mistaken by the immune system for opsonized viruses, resulting in the production of type I interferons, a hallmark of SLE (pathogenesis). The pathogenicity of autoantibodies is thought to strongly increase if autoantigens are accessible for immune-complex formation. The immune complex could be considered a binary pyrogen formed from less proinflammatory components. The accessibility of cognate autoantigens, in turn, is likely to be related to impaired or delayed clearance of apoptotic cells.

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.

Macrophages Discriminate Glycosylation Patterns of Apoptotic Cell-derived Microparticles

Background: Apoptotic cells release vesicles, which expose “eat-me” signals. Results: Vesicles originated from endoplasmic reticulum expose immature glycoepitopes and are preferentially phagocytosed by macrophages. Conclusion: Immature surface glycoepitopes serve as “eat-me” signals for the clearance of apoptotic vesicles originated from endoplasmic reticulum. Significance: Understanding the distinction by macrophages of apoptotic blebs may provide new insights into clearance-related diseases. Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.

Hypercoagulability Inhibits Monocyte Transendothelial Migration Through Protease-Activated Receptor-1-, Phospholipase-Cβ-, Phosphoinositide 3-Kinase-, and Nitric Oxide-Dependent Signaling in Monocytes and Promotes Plaque Stability

Background— Clinical studies failed to provide clear evidence for a proatherogenic role of hypercoagulability. This is in contrast to the well-established detrimental role of hypercoagulability and thrombin during acute atherosclerotic complications. These seemingly opposing data suggest that hypercoagulability might exert both proatherogenic and antiatherogenic effects. We therefore investigated whether hypercoagulability mediates a beneficial effect during de novo atherogenesis. Methods and Results— De novo atherogenesis was evaluated in 2 mouse models with hyperlipidemia and genetically imposed hypercoagulability (TMPro/ProApoE−/− and FVLQ/QApoE−/− mice). In both mouse models, hypercoagulability resulted in larger plaques, but vascular stenosis was not enhanced secondary to positive vascular remodeling. Importantly, plaque stability was increased in hypercoagulable mice with less necrotic cores, more extracellular matrix, more smooth muscle cells, and fewer macrophages. Long-term anticoagulation reversed these changes. The reduced frequency of intraplaque macrophages in hypercoagulable mice is explained by an inhibitory role of thrombin and protease-activated receptor-1 on monocyte transendothelial migration in vitro. This is dependent on phospholipase-Cβ, phosphoinositide 3-kinase, and nitric oxide signaling in monocytes but not in endothelial cells. Conclusions— Here, we show a new function of the coagulation system, averting stenosis and plaque destabilization during de novo atherogenesis. The in vivo and in vitro data establish that thrombin-induced signaling via protease-activated receptor-1, phospholipase-Cβ, phosphoinositide 3-kinase, and nitric oxide in monocytes impairs monocyte transendothelial migration. This likely accounts for the reduced macrophage accumulation in plaques of hypercoagulable mice. Thus, in contrast to their role in unstable plaques or after vascular injury, hypercoagulability and thrombin convey a protective effect during de novo atherogenesis.

Cholera toxin binds to lipid rafts but has a limited specificity for ganglioside GM1.

Lipid rafts and the formation of an immunological synapse are crucial for T‐cell activation. Binding of cholera toxin B subunit (CTB) to ganglioside GM1 is a marker to identify lipid rafts. Primary human T cells were isolated from healthy donors and were stimulated with superantigen staphylococcus enterotoxin B (SEB) and stained with cholera toxin B‐fluorescein isothiocyanate (CTB‐FITC). An optimized staining procedure is required to stain lipid rafts exclusively on the cell surface. Unstimulated T cells show a few CTB binding spots on the cell surface. The size and number of CTB‐binding lipid rafts are strongly upregulated during T‐cell activation in SEB‐stimulated CD4+ T cells. However, our data show that the specificity of CTB for GM1 ganglioside is limited, because the binding capacity is partly resistant to inhibition of ganglioside synthesis and sensitive to trypsin digestion. Our results indicate that the binding of FITC‐labeled CTB can be divided into at least three different categories: a specific binding of CTB to ganglioside GM1, a nonspecific binding of CTB probably to glycosylated surface proteins and a nonspecific binding of FITC to the cell surface.

Atorvastatin Inhibits T Cell Activation through 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase without Decreasing Cholesterol Synthesis

The localization of the TCR and other signaling molecules in membrane rafts (MR) is essential for the activation of T lymphocytes. MR are stabilized by sphingolipids and cholesterol. Activation of T lymphocytes leads to the confluence of small MR and the formation of an immunological synapse that is essential for sustained activation and proliferation. In this study, we investigated the effect of statins on MR and T cell activation in superantigen-stimulated human PBMC. Atorvastatin significantly inhibited cellular activation and proliferation. The binding of cholera toxin B subunit to isolated MR and to whole cells was inhibited by low doses of statins. Statins reduce the association of critical signaling proteins such as Lck and linker of activation in T cells with MR in stimulated T cells. The expression of activation markers CD69 and CD25 was inhibited. Several statin-mediated mechanisms, such as a lower stimulation with MHC-II, an inhibition of costimulation by direct binding of statins to LFA-1, a reduced secretion of cytokines, or a depletion of cellular cholesterol pools, were excluded. Inhibition of protein prenylation had a similar effect on T cell proliferation, suggesting that a reduced protein prenylation might contribute to the statin-mediated inhibition of T cell activation. Statins induce both lower levels of low-density lipoprotein cholesterol and inhibition of T cell activation, which might contribute to an inhibition of atherosclerosis.

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.

Enhanced killing of B lymphoma cells by granulocyte colony‐stimulating factor‐primed effector cells and Hu1D10 – a humanized human leucocyte antigen DR antibody

Summary. Antibody‐based approaches have become a novel treatment modality for lymphoma patients. Humanized 1D10 (Hu1D10; Remitogen) is among the antibodies that are currently under evaluation in phase II clinical trials in lymphoma patients. The 1D10 antibody is directed against a polymorphic epitope on the β‐chain of human leucocyte antigen (HLA) class II. We found expression of the 1D10 epitope on B cells and monocytes from approximately 50% of healthy donors. Analyses of 1D10 expression on malignant cells revealed that approximately half of the HLA class II‐positive haematological malignancies expressed the 1D10 epitope. In whole blood antibody‐dependent cellular cytotoxicity (ADCC) assays, Hu1D10 was more effective than rituxan in killing malignant ARH‐77 B cells. Interestingly, Hu1D10‐mediated lymphoma cell lysis was significantly enhanced when blood from granulocyte colony‐stimulating factor (G‐CSF)‐treated patients was compared with blood from healthy controls. Analyses of the relevant effector cell populations revealed that FcγRI (CD64)‐positive polymorphonuclear cells were critical for enhanced Hu1D10‐mediated lymphoma killing during G‐CSF therapy, while the same effector cell population induced only marginal lysis with rituxan. Furthermore, Hu1D10 was highly effective in inducing apoptosis in primary lymphoma cells from B chronic lymphocytic leukaemia patients. These preclinical results form the basis for a phase I/II clinical trial of Hu1D10 in combination with G‐CSF.

Pathogen-Triggered Activation of Plasmacytoid Dendritic Cells Induces IL-10–Producing B Cells in Response to Staphylococcus aureus

Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell–independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the VH3+ BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A–bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scidPrkdc/γc−/− mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell–derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell–mediated immune tolerance.