Secondary Necrotic Cells Research Articles

TNF/TNF-R1 pathway is involved in doxorubicin-induced acute sterile inflammation

Dear Editor, Doxorubicin is an anthracycline antibiotic, which is used in treatment of cancer and it works by induction of apoptosis in cancerous cells.1 In vivo, apoptotic cells are rapidly cleared preferentially by monocytes and neutrophil migration is inhibited2 to limit tissue injury and inflammation. More recently, few anti-cancer drugs, including anthracyclines (doxorubicin) were shown to evoke apoptosis in cancerous cells, which is associated with immune system activation (i.e. immunogenic apoptosis).3, 4 In our previous study we reported that doxorubicin can induce acute inflammation in peritoneum, which is associated with apoptosis.5 Doxorubicin-killed cells could be also a source of damage-associated molecular patterns (DAMPs) leading to inflammation and tumor necrosis factor (TNF) production, which would amplify the inflammatory response triggered by DAMPs. TNF is a pleiotropic cytokine that binds to receptors TNF-R1 and TNF-R2 and, depending on cell type, triggers different signaling pathways, including cell death and inflammation.6 This study aimed to examine whether TNF contributes to the doxorubicin-induced acute sterile inflammation. Intraperitoneal injection of doxorubicin provoked an acute inflammatory response accompanied by the influx of neutrophils.5 We observed an increased level of LDH, a marker of tissue damage and secondary necrotic cells, already after 6 h with further increase after 16 h post injection of doxorubicin (Supplementary Figure 1a). Lavage fluid collected 16 h after doxorubicin injection contained increased levels of TNF as compared to vehicle-injected group (0.09±0.14 versus 2.11±0.84 pg/ml, P<0.0001). We next tested the involvement of TNF-R1 and TNF-R2 in the sterile inflammation in response to doxorubicin. Wild-type mice had abundant neutrophils in their abdominal cavities, but this response was markedly decreased in TNF-R1/2 double knockout mice (by 2.4-fold, Supplementary Figure 1b). The number of neutrophils attracted in response to doxorubicin-induced acute inflammation was 3.4-fold lower in TNF-R1-deficient mice than in controls (Figure 1a), but in TNF-R2-deficient mice, there was no difference as compared to wild-type mice (Figure 1b). These results show that TNF-R1 was involved in mediating the inflammatory response but TNF-R2 was not. Importantly, cell death in TNF-R1/2 double knockout mice was not affected in comparison to wild-type mice (data not shown) indicating that TNF does not contribute to the cell death process itself. Figure 1 Role of TNF-R1 and TNF-R2 and the effect of inhibiting soluble TNF on doxorubicin-induced acute inflammatory response. The number of neutrophils in peritoneal lavage was decreased in TNF-R1−/− (a) but not in TNF-R2−/− ( ... Further, we investigated whether acute inflammation can be reduced in wild-type mice by the administration of Etanercept, a recombinant dimeric soluble form of TNF-R2 that blocks the interaction of TNF with its cell surface receptors.7 Wild-type mice were injected intraperitoneally with Etanercept together with doxorubicin and 16 h later the recruited cells were phenotyped. Etanercept significantly reduced the recruitment of neutrophils (Figure 1c). This further confirms the importance of TNF in doxorubicin-induced acute inflammation. In conclusion, we report that intraperitoneal injection of doxorubicin in mice leads to increase in TNF levels in the lavage fluid. It has been also reported that patients undergoing chemotherapy with doxorubicin had elevated levels of TNF in their plasma.8 Similarly, mouse in vitro and in vivo studies demonstrated up-regulation of TNF after doxorubicin administration both on mRNA and protein levels.9, 10 Previously, we found that the majority of cells that died apoptotically due to doxorubicin treatment were monocytes/macrophages with some minor neutrophils.5 Therefore, it is possible that either dying macrophages or attracted neutrophils might be a source of TNF, and that by binding to TNF-R1 it could amplify the inflammation. We demonstrated that the acute inflammatory response to doxorubicin was reduced in TNF-R1- but not TNF-R2-deficient mice. In addition, Etanercept decreases the attraction of neutrophils after doxorubicin administration. These studies show that TNF and the TNF-R1 signaling pathway are key elements in the acute sterile inflammatory response to doxorubicin.

Dendritic cells generated with Flt3L and exposed to apoptotic cells lack induction of T cell anergy and Foxp3+ regulatory T cell conversion in vitro

Removal of apoptotic cells, which appear during the steady state, is a pre-requisite to prevent generation of secondary necrotic cells that may lead to autoimmunity. The recognition of apoptotic material by dendritic cells (DCs) has been proposed to convert them into tolerogenic DCs equipped with specialized tolerogenic mechanisms on T cells. However, comparative studies to demonstrate functional alterations of DCs upon exposure to apoptotic cells have not been performed so far. Here we show that immature murine bone marrow-derived DCs generated with GM-CSF (GM-DCs) or Flt3L (FL-DCs) interact with live or apoptotic syngeneic thymocytes. As expected, GM-DCs phagocytose apoptotic but not live cells, FL-DCs only show trogocytosis of membrane parts. Interaction with live or apoptotic thymocytes did not lead to DC maturation. Both GM-DCs and FL-DCs present OVA as protein, peptide and membrane-associated antigens. Interestingly, only GM-DCs were able to induce T cell anergy or convert naïve T cells into FoxP3+ regulatory T cells (Tregs) but FL-DCs did not show either of these effects. Unexpectedly, exposure of immature GM-DCs to live or apoptotic thymocytes did not improve DC functions in both types of in vitro T cell tolerance induction assays. Together, our data suggest that these tolerogenic in vitro measures of immature BM-DCs are not further enhanced by exposure to apoptotic cells and may depend on the generating cytokine.

SAT0162 Complexes of anti-CRP autoantibodies and CRP-opsonised apoptotic cell remnants form a pro-inflammatory pathogen in systemic lupus erythematosus

BackgroundAn efficient clearance of dying and dead cells is essential to avoid inflammation and autoimmunity. Therefore, a plethora of opsonizing molecules secures silent clearance [1]. Opsonisation with C-reactive protein (CRP)...

AB0208 Immune complex formation after exposure of autoantigens on the surface of secondary necrotic cells (SNEC) promotes inflammation in SLE

BackgroundSystemic Lupus Erythematosus (SLE) is a chronic inflammatory disease characterised by the production of autoantibodies, formation of immune complexes (IC), and activation of complement.[1] Accumulation in blood and tissues of...

Colourful death: Six-parameter classification of cell death by flow cytometry—Dead cells tell tales

The response of the immune system against dying and dead cells strongly depends on the cell death phenotype. Beside other forms of cell death, two clearly distinct populations, early apoptotic and secondary necrotic cells, have been shown to induce anti-inflammation/tolerance and inflammation/immune priming, respectively. Cytofluorometry is a powerful technique to detect morphological and phenotypical changes occurring during cell death. Here, we describe a new technique using AnnexinA5, propidiumiodide, DiIC1(5) and Hoechst 33342 to sub-classify populations of apoptotic and/or necrotic cells. The method allows the fast and reliable identification of several different phases and pathways of cell death by analysing the following cell death associated changes in a single tube: cellular granularity and shrinkage, phosphatidylserine exposure, ion selectivity of the plasma membrane, mitochondrial membrane potential, and DNA content. The clear characterisation of cell death is of major importance for instance in immunization studies, in experimental therapeutic settings, and in the exploration of cell-death associated diseases. It also enables the analysis of immunological properties of distinct populations of dying cells and the pathways involved in this process.

Heparin can liberate high molecular weight DNA from secondary necrotic cells

The borderline between necrosis and apoptosis is indistinct, but that between types of cell death is important because necrosis may lead to local inflammation, whereas apoptosis usually does not. In certain autoimmune disorders, inhibition of cell death is crucial, since macromolecules released from the dead cells may accelerate the autoimmune processes. We have used various cell death inhibitors to block cell death induced by 4HPR [N-(4-hydroxyphenil)-retinamide] the BL41 and U937 cell lines. VD-FMK, a general caspase inhibitor, inhibited DNA fragmentation induced by 4HPR, but not PI (propidium iodide) uptake and necrosis. Interestingly heparin, a serine-protease inhibitor, lowered the PI fluorescence of the dead cell population and increased the sub-G1 population as measured by flow cytometry. Regarding these changes, we found that heparin failed to increase DNA fragmentation, but merely liberated high molecular mass DNA fragments from dead cells. The exact mechanism is unclear, but heparin during secondary necrosis might enter the cells, bind RNPs (ribonucleoproteins), and pull them out with the attached DNA, where they would be sensitive to enzymatic degradation. Thus, the results suggest that heparin treatment helps in the clearance of cell debris and decreases the immunogenity of secondary necrotic cells.

Immune complex formation after exposure of autoantigens on the surface of secondary necrotic cells (SNEC) promotes inflammation in SLE

Background and objectivesSystemic lupus erythematosus (SLE) is a chronic inflammatory disease characterised by the production of autoantibodies, formation of immune complexes (IC), and activation of complement. Accumulation in blood and...

Corynebacterium diphtheriae 67-72p hemagglutinin, characterized as the protein DIP0733, contributes to invasion and induction of apoptosis in HEp-2 cells

Although Corynebacterium diphtheriae has been classically described as an exclusively extracellular pathogen, there is growing evidence that it may be internalized by epithelial cells. The aim of the present report was to investigate the nature and involvement of the surface-exposed non-fimbrial 67–72 kDa proteins (67-72p), previously characterized as adhesin/hemagglutinin, in C. diphtheriae internalization by HEp-2 cells. Transmission electron microscopy and bacterial internalization inhibition assays indicated the role of 67-72p as invasin for strains of varied sources. Cytoskeletal changes with accumulation of polymerized actin in HEp-2 cells beneath adherent 67-72p-adsorbed microspheres were observed by the Fluorescent actin staining test. Trypan blue staining method and Methylthiazole tetrazolium reduction assay showed a significant decrease in viability of HEp-2 cells treated with 67-72p. Morphological changes in HEp-2 cells observed after treatment with 67-72p included vacuolization, nuclear fragmentation and the formation of apoptotic bodies. Flow cytometry revealed an apoptotic volume decrease in HEp-2 cells treated with 67-72p. Moreover, a double-staining assay using Propidium Iodide/Annexin V gave information about the numbers of vital vs. early apoptotic cells and late apoptotic or secondary necrotic cells. The comparative analysis of MALDI-TOF MS experiments with the probes provided for 67-72p CDC-E8392 with an in silico proteome deduced from the complete genome sequence of C. diphtheriae identified with significant scores 67-72p as the protein DIP0733. In conclusion, DIP0733 (67-72p) may be directly implicated in bacterial invasion and apoptosis of epithelial cells in the early stages of diphtheria and C. diphtheriae invasive infection.

Macrophages in inflammation and its resolution

Macrophages are highly plastic leukocytes that differentiate from monocytes following their entry into extravascular tissues. Macrophages can enter various tissues under inflammatory or non-inflammatory conditions and assume different functions and phenotypes according to the cues they receive from the environment. The notion that inflammation in general and macrophage responses in particular affect physiological phenomena that were previously considered to be not immune-related has enhanced and broadened our understanding of macrophage function during inflammation and its resolution. This volume brings together 14 manuscripts that cover various aspects of macrophage function during inflammation and its resolution, as well as in several pathologic states for which a significant, long-lasting, macrophage-mediated immune response plays a significant role. Two of the manuscripts present original research on macrophage phagocytosis and its implications. Five provide an overview of macrophage function during inflammation and its resolution, with an emphasis on the modulatory role of particular elements in this response, such as apoptotic leukocytes, specific pathogens, hypoxia, and hormone receptors. The remaining seven manuscripts outline the role of macrophages during inflammation and its resolution in different tissues, including the lung, cardiovascular and adipose tissues, injured skeletal muscle and neuronal tissues, and synovial and oral cavities. The two original research articles are devoted to the consequences of particle engulfment by macrophages. Labrousse et al. (2011) describe a novel experimental strategy in which they use micro-patterned immune complexes to trigger frustrated phagocytosis and thereby determine spatial parameters in lysosomal movement and fusion. Janko et al. (2011) report on the cumulative binding of CRP and anti-CRP antibodies to the surface of secondary necrotic cells. This binding leads to a pro-inflammatory cytokine response following engulfment by macrophages, implying a potential role for these elements in the etiology of systemic lupus erythematosus. Of the review articles that discuss the regulation of macrophage differentiation and function by discrete events, two cover the interaction between macrophages and apoptotic leukocytes during the resolution of inflammation. Korns et al. (2011) outline the regulation of apoptotic cell clearance by macrophages (efferocytosis) and the environmental cues that promote the efferocytic capabilities of macrophages. The second manuscript by Ariel and Serhan (2012) reviews the impact of apoptotic cell sensing and disposal by macrophages on the switches in functional phenotypes displayed by these cells. The effect of another environmental factor, hypoxia, on monocyte/macrophage activation, and differentiation through transcriptional and translational modulation is covered by Rahat et al. (2011). Lugo-Villarino et al. (2011) discuss the pathogenesis and co-mortality displayed by two macrophage-inhabiting microbes (HIV and Mycobacterium Tuberculosis) and their influence on macrophage polarization. Patel et al. (2011) review the role of melanocortin receptor expression by macrophages in anti-inflammation and the resolution of inflammation, with attention given to melanocortin receptor agonists as therapeutic agents. Several review articles discuss the function of macrophages during inflammation and/or its resolution within distinct anatomical sites, taking into account the unique features of these tissue-specific macrophages, in particular the distinct environments in which they reside and their interactions with neighboring cells. Claria et al. (2011) review current knowledge on the contribution of macrophages to the inflammatory state characterizing adipose tissue and the phenotypic changes observed in macrophages during obesity. Kennedy et al. (2011) discuss macrophage polarization occurring within the synovial space of arthritic joints and its modulation by cytokines, transcription factors, and pro-resolving lipid mediators. The article from Bosurgi et al. (2011) describes the multiple actions of macrophages in injured skeletal muscle, where the effects of these cells are a double-edged sword and can either promote healing and repair or lead to fibrosis and fat replacement. Herold et al. (2011) survey the indispensable role of macrophages in the resolution and termination of inflammation in lung infection and injury as well as the molecular pathways involved in these processes. Proper termination of inflammatory events and clearance of apoptotic cells are also critical to the cardiovascular system, as reviewed by Thorp (2012), and defects in macrophage efferocytosis can lead to atherosclerosis and myocardial infarction. While monocyte-derived macrophages and resident microglia were previously considered to be detrimental in brain inflammation and injury, recent advances reviewed by Jung and Schwartz (2012) suggest an opposite role for these macrophage-like cells, with a positive impact on brain maintenance and repair. Finally, Hasturk et al. (2012) outline the reciprocal interaction between periodontal disease and chronic inflammatory illnesses and the role that macrophages play in mediating these chronic inflammatory diseases. Altogether, the articles in this volume portray the complexity of the multiple roles played by macrophages and members of their lineage during inflammation and its resolution, and their manipulation by the injured milieu. These topics are currently heavily studied, and advances in the field, facilitated by state-of-the-art genetics and optical technologies, will undoubtedly continue to contribute to our understanding of the immune system's response to foreign insults, trauma, and inflammatory disorders.

DAMPs and PDT-mediated photo-oxidative stress: exploring the unknown

Damage-associated molecular patterns (DAMPs) or cell death associated molecular patterns (CDAMPs) are a subset of endogenous intracellular molecules that are normally hidden within living cells but become either passively released by primary and secondary necrotic cells or actively exposed and secreted by the dying cells. Once released, DAMPs are sensed by the innate immune system and act as activators of antigen-presenting cells (APCs) to stimulate innate and adaptive immunity. Cancer cells dying in response to a subset of conventional anticancer modalities exhibit a particular composition of DAMPs at their cell surface, which has been recently shown to be vital for the stimulation of the host immune system and the control of residual disease. Photodynamic therapy (PDT) for cancer has long been shown to be capable of killing malignant cells and concomitantly stimulate the host immune system, properties that are likely linked to its ability of inducing exposure/release of certain DAMPs. PDT, by evoking oxidative stress at specific subcellular sites through the light activation of organelle-associated photosensitizers, may be unique in incorporating tumour cells destruction and antitumor immune response in one therapeutic paradigm. Here we review the current knowledge about mechanisms and signalling cascades leading to the exposure of DAMPs at the cell surface or promoting their release, the cell death mechanism associated to these processes and its immunological consequences. We also discuss how certain PDT paradigms may yield therapies that optimally stimulate the immune system and lead to the discovery of new DAMPs.

CRP discriminates primary from secondary necrosis

Background and objectivesC-reactive protein (CRP) is an acute-phase protein whose serum concentration increases massively in response to tissue injury or inflammation. Beside membranes of bacteria CRP is known to bind...

CRP/anti-CRP Antibodies Assembly on the Surfaces of Cell Remnants Switches Their Phagocytic Clearance Toward Inflammation

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease characterized by the production of autoantibodies, formation of immune complexes (IC), and activation of complement that ultimately fuel acute and/or chronic inflammation. Accumulation in blood and tissues of post-apoptotic remnants is considered of etiological and pathological importance for patients with SLE. Besides receptors directly recognizing apoptotic cells, soluble opsonins of the innate immune system bind apoptotic material dependent on the stage of apoptosis. We describe the binding to the surface of secondary necrotic cells (SNEC) of the serum opsonin CRP and further opsonins. We show that anti-dsDNA and anti-CRP autoantibodies bind and sensitize SNEC. Autoantibody-sensitized SNEC were cleared by macrophages in vitro and induced a pro-inflammatory cytokine response. In conclusion, anti-CRP, CRP, and SNEC form a ternary pyrogen endowed with strong pro-inflammatory capabilities which is able to maintain and perpetuate chronic inflammation.

Natural IgM and innate immune collectin SP-D bind to late apoptotic cells and enhance their clearance by alveolar macrophages in vivo

Innate immune collectin surfactant protein D (SP-D) and natural immunoglobulin M (IgM) are two soluble proteins. These opsonic proteins are good candidates for enhancing late apoptotic cell clearance. However, effects of these proteins on late apoptotic cell clearance in the lungs are not clearly established. We have recently shown that SP-D can bind several immunoglobulin isotypes, including IgM. Here we hypothesized that IgM and SP-D bind to late apoptotic cells and enhance their clearance from the lungs. We show that IgM and SP-D bind to late apoptotic secondary necrotic cells, and that IgM and SP-D either co-localize to the same regions or to different regions of late apoptotic Jurkat T cells. Mouse alveolar macrophages internalized late apoptotic cells, in vivo. We induced lung inflammation in mice using LPS and show that airway IgM and SP-D levels and the clearance of late apoptotic cells by alveolar macrophages increases under these conditions. We then coated late apoptotic cells with IgM, SP-D, or both and instilled them into the mouse airways. We found that alveolar macrophages internalize IgM- and SP-D-coated late apoptotic cells more effectively than uncoated late apoptotic cells, in vivo. None of these conditions cause inflammation in the naïve lungs. Therefore, these data suggest that both IgM and SP-D effectively opsonize late apoptotic cells and directly enhance their clearance by alveolar macrophages in the lungs.

Toxicity of the ichthyotoxic marine flagellate Pseudochattonella (Dictyochophyceae, Heterokonta) assessed by six bioassays

Recurrent blooms of the marine flagellate genus Pseudochattonella have caused several fish kills in Northern Europe and Japan during the last two decades. Six different bioassays using fish cells, fish and brine shrimp nauplii were assessed as test systems for investigating ichthyotoxic mechanisms of Pseudochattonella. Metabolic activity in embryo cells from chinook salmon (CHSE-214) was inhibited in 2 strains, increased in another strain but was unaffected in a further 2 strains when exposed to extracts from strains of Pseudochattonella farcimen while it increased after exposure to both strains of Pseudochattonella verruculosa. Primary hepatocyte cells from rainbow trout showed metabolic inhibition and loss of membrane integrity when exposed to DMSO extracts both from Pseudochattonella spp. and from the putatively non-toxic heterokonts Pseudopedinella pyriforme and Skeletonema sp. while the same algae extracted with H 2O showed no adverse effects in the bioassays used. No acute mortality was observed after 24 h exposure of cod fry and salmon smolts to Pseudochattonella and Pseudopedinella. However, histological examinations of fish gills showed strong effects on secondary lamellae (hypertrophy, edema, thickened secondary lamellae and necrotic cells). No acute toxicity of brine shrimp larvae could be detected after 24 h exposure to any of the Pseudochattonella strains. This study shows that both Pseudochattonella and Pseudopedinella affect cell metabolism and can cause damage to gills. The toxicity of Pseudochattonella varied by species, strain and bioassay system, and was most clearly demonstrated when salmon smolts or cod fry were exposed to bloom concentrations of P. verruculosa from Japan.

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.

Dangerous attraction: phagocyte recruitment and danger signals of apoptotic and necrotic cells

Tissue homeostasis in metazoa requires the rapid and efficient clearance of dying cells by professional or semi-professional phagocytes. Impairment of this finely regulated, fundamental process has been implicated in the development of autoimmune diseases, such as systemic lupus erythematosus. Various studies have provided us a detailed understanding of the interaction between dying cells and phagocytes as well as the current concept that apoptotic cell removal leads to a non- or anti-inflammatory response, whereas necrotic cell removal stimulates a pro-inflammatory reaction. In contrast, our knowledge about the soluble factors released from dying cells is rather limited, although meanwhile it is generally accepted that not only the dying cell itself but also the substances liberated during cell death contribute to the process of corpse clearance and the subsequent immune response. This review article is intended as an up-to-date survey over attraction and danger signals of apoptotic, primary and secondary necrotic cells, their function as chemoattractants in phagocyte recruitment, additional effects on the immune system, and the receptors, which are engaged in this scenario.

Cell Surface Externalization of Annexin A1 as a Failsafe Mechanism Preventing Inflammatory Responses during Secondary Necrosis

The engulfment of apoptotic cells is of crucial importance for tissue homeostasis in multicellular organisms. A failure of this process results in secondary necrosis triggering proinflammatory cytokine production and autoimmune disease. In the present study, we investigated the role of annexin A1, an intracellular protein that has been implicated in the efficient removal of apoptotic cells. Consistent with its function as bridging protein in the phagocyte synapse, opsonization of apoptotic cells with purified annexin A1 strongly enhanced their phagocytic uptake. A detailed analysis, however, surprisingly revealed that annexin A1 was hardly exposed to the cell surface of primary apoptotic cells, but was strongly externalized only on secondary necrotic cells. Interestingly, while the exposure of annexin A1 failed to promote the uptake of these late secondary necrotic cells, it efficiently prevented induction of cytokine production in macrophages during engulfment of secondary necrotic cells. Our results therefore suggest that annexin A1 exposure during secondary necrosis provides an important failsafe mechanism counteracting inflammatory responses, even when the timely clearance of apoptotic cells has failed.

AnnexinA5 renders dead tumor cells immunogenic—implications for multimodal cancer therapies

Multimodal tumor therapies should aim not only to kill the tumor cells, but also to stimulate a specific immune response to keep residual tumor (stem) cells and metastases under control. Apoptotic cells are mostly noninflammatory or even anti-inflammatory while necrotic cells stimulate the immune system. Whether the immunogenicity of apoptotic tumor cells can be increased by interfering with their swift and phosphatidylserine (PS)-dependent clearance by macrophages was examined. AnnexinA5 (AnxA5) is a naturally occurring highly specific ligand for PS. Proteins of the annexin family are characterized by a selective affinity for phospholipids in the presence of Ca2+ ions. The phagocytosis by macrophages of irradiated, apoptotic tumor cells (ITC) was partially inhibited when the ITC were preincubated with AnxA5. Activated macrophages secreted higher amounts of TNFα and IL-1β after contact with ITC plus AnxA5 in comparison with ITC alone, while the amount of TGF-β was decreased. Macrophages of AnxA5-deficient mice showed an increased phagocytosis of dead cells. Wild-type mice, where endogenous AnxA5 is present, displayed a significantly faster decline in size of allogeneic tumors in comparison with AnxA5-deficient animals. The addition of AnxA5 to ITC vaccines increased the percentage of tumor-free mice in syngeneic tumor protection and tumor cure assays. AnxA5 alone led to a retard of syngeneic tumor growth that was, however, less pronounced in comparison to treatment of the tumor with ionizing irradiation. In conclusion, AnxA5 disturbs the PS-dependent clearance by macrophages of dying tumor cells, leading to the accumulation of the latter, to the occurrence of secondary necrotic cells, and to an increased uptake of the dead cells by dendritic cells. Tumor cure appendages with dead tumor cells should be performed with AnxA5 as an immune stimulator and could be combined with irradiation, chemotherapy, and hyperthermia to induce immunogenic cell death forms in vivo or ex vivo.

Altered expression of glycoproteins on the cell surface of Jurkat cells during etoposide-induced apoptosis: Shedding and intracellular translocation of glycoproteins

Background The glycoproteins on the cell surface are altered during apoptosis and play an important role in phagocytic clearance of apoptotic cells. Methods We classified Jurkat cells treated with etoposide as viable and early apoptotic cells, late apoptotic cells or secondary necrotic cells based on propidium iodide staining and scattered grams and estimated the expression levels of glycoproteins on the cell surface. Results The cell surface expression levels of intercellular adhesion molecules (ICAM)-2 and -3 on the apoptotic cells were markedly lower, while those of calnexin, calreticulin, and lysosome-associated membrane proteins (LAMP)-1 and -2 were significantly higher compared to non-apoptotic cells. These decreases in ICAM-2 and -3 on the apoptotic cell surface were reduced in the presence of metalloproteinase inhibitors and caspase inhibitors, respectively. Confocal microscopic analysis revealed that calnexin and calreticulin were assembled around fragmented nuclei of blebbed apoptotic cells. Conclusions These results suggest that alteration of glycoproteins on the cell surface during apoptosis is associated with shedding and intracellular translocation of glycoproteins.

Complement classical pathway components are all important in clearance of apoptotic and secondary necrotic cells

Inherited deficiencies in components of the classical complement pathway are strong disease susceptibility factors for the development of systemic lupus erythematosus (SLE) and there is a hierarchy among deficiency states, the strongest association being with C1q deficiency. We investigated the relative importance of the different complement pathways regarding clearance of apoptotic cells. Phagocytosis of labelled apoptotic Jurkat cells by monocyte-derived macrophages in the presence of sera from individuals with complement deficiencies was studied, as well as C3 deposition on apoptotic cells using flow cytometry. Sera from individuals deficient in C1q, C4, C2 or C3 all showed decreased phagocytosis. Mannose binding lectin (MBL) and the alternative pathway did not influence phagocytosis. Notably, the components of the complement classical pathway, including C1q, were equally important in clearance of apoptotic cells. This indicates that deposition of C3 fragments is of major significance; we therefore studied C3 deposition on apoptotic cells. Experiments with MBL-deficient serum depleted of C1q or factor D confirmed the predominance of the classical pathway. At low dilution, sera deficient of C1q, C4 or C2 supported C3 fragment deposition demonstrating alternative pathway activation. In conclusion, we have found that complement-mediated opsonization and phagocytosis of apoptotic cells, particularly those undergoing secondary necrosis, are dependent mainly upon an intact classical pathway. The alternative pathway is less important, but may play a role in some conditions. C1q was not more important than other classical pathway components, suggesting a role in additional pathogenetic processes in SLE other than clearance of apoptotic cells.