No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Inefficient Clearance of Dying Cells and Autoreactivity
Abstract
Dying cells were basically unnoticed by scientists for a long time and only came back into the spotlight roughly 10 years ago. The process of recognition and uptake of apoptotic and necrotic cells is complex and failures in this process can contribute to the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Here, we discuss the recognition and uptake molecules which are involved in an efficient clearance of dying cells in early and late phases of cell death. The exposure of phosphatidylserine (PS) is an early surface change of apoptosing cells recognized by several receptors and adaptor molecules. We demonstrated that dying cells have cell membranes with high lateral mobility of PS, which contribute to their efficient clearance. Changes of the glycoprotein composition of apoptotic cells occur later than the exposure of PS. We further observed that complement binding is an early event in necrosis and a rather late event in apoptosis. Complement, C-reactive protein (CRP), and serum DNase I act as back-up molecules in the clearance process. Finally, we discuss how the accumulation of secondary necrotic cells and cellular debris in the germinal centers of secondary lymph organs can lead to autoimmunity. It is reasonable to argue that clearance defects are major players in the development of autoimmune diseases such as SLE.
... tumorképződés) [19], nem pótlódnak (pl. a szövet öregedése), gyulladás vagy autoimmun reakció jelentkezik (pl. elhalt sejtek felhalmozódásakor) [20,21,22,23]. ...
... Kiléphetnek a véráramból, és különböző szövetekben rögzülhetnek mint szöveti makrofágok. A szöveti makrofágok típusai: histiocyták a lazarostos kötőszövetben, KUPFFER 19 -sejtek a májban, osteoclastok a csontszövetben (a többi makrofággal ellentétben nem fagocitálnak, de velük közös sejtvonalból származnak), chondroclastok a porcszövetben [95] 20 vérében található KURLOFF-sejt 21 [92,97]. ...
... A tengerimalacfélék közé tartozik, Dél-Amerikában honos. 21 MIHAIL KURLOFF (1859-1932) orosz orvos. A KURLOFF-sejt lymphocyta és monocyta tulajdonságokat egyesítő sejt, in vitro természetes ölősejtként viselkedik. ...
University lecture book in Hungarian. 1st edition 2005, 2nd, revised edition 2012.
... Lupus nephritis is a serious manifestation of Systemic lupus erythematosus (SLE) and a major predictor of poor outcome [1,2]. The predominance of chromatin-associated autoantigens involved in lupus nephritis points at deficiencies in the processing and clearance of chromatin from dead cells as central factors in the pathogenesis of SLE [3][4][5][6][7][8]. Enzymatic DNA fragmentation by different endonucleases is significant during apoptotic cell death (reviewed in [9,10]), and in the elimination of chromatin released from necrotic cells (reviewed in [9][10][11]). ...
... This appears when mild mesangial lupus nephritis transforms into end-stage organ disease [5]. Without adequate degradation by DNaseI, chromatin may transform into secondary necrotic chromatin released from apoptotic blebs [7,8,14,15]. In this situation, chromatin fragments may exert central roles in the pathogenesis of SLE. ...
... Such cytokines can directly up-regulate MMPs [22,25,26,28,29]. Alternatively, incomplete clearance and degradation of apoptotic cells may transform them into secondary necrotic cell debris [5,7,[30][31][32]. Necrotic cell debris contains SAP130, which serves as a ligand for the inflammation-related receptor Clec4e [33]. ...
Recent studies demonstrate that transformation of mild lupus nephritis into end-stage disease is imposed by silencing of renal DNaseI gene expression in (NZBxNZW)F1 mice. Down-regulation of DNaseI results in reduced chromatin fragmentation, and in deposition of extracellular chromatin-IgG complexes in glomerular basement membranes in individuals that produce IgG anti-chromatin antibodies. The main focus of the present study is to describe the biological consequences of renal DNaseI shut-down and reduced chromatin fragmentation with a particular focus on whether exposed large chromatin fragments activate Toll like receptors and the necrosis-related Clec4e receptor in murine and human lupus nephritis. Furthermore, analyses where performed to determine if matrix metalloproteases are up-regulated as a consequence of chromatin-mediated Toll like receptors/Clec4e stimulation. Mouse and human mRNA expression levels of DNaseI, Toll like receptors 7-9, Clec4e, pro-inflammatory cytokines and MMP2/MMP9 were determined and compared with in situ protein expression profiles and clinical data. We demonstrate that exposure of chromatin significantly up-regulate Toll like receptors and Clec4e in mice, and also but less pronounced in patients with lupus nephritis treated with immunosuppresants. In conclusion, silencing of renal DNaseI gene expression initiates a cascade of inflammatory signals leading to progression of both murine and human lupus nephritis. Principal component analyses biplot of data from murine and human lupus nephrits demonstrate the importance of DNaseI gene shut down for progression of the organ disease.
... Cells dying by autophagic cell death (autophagy fails and induces apoptosis) seem to behave similar to normal apoptotic cells [94]. Necrotic cells should be divided into secondary and primary necrotic cells (for extensive review, see [63]). Necrotic cells are characterized by disturbed membrane integrity. ...
... These MP are released very early during the apoptotic process and are swiftly cleared by macrophages. If clearance of apoptotic cells is delayed or inhibited (for review, see [55,62,63]), the apoptotic cell collapses and fragments. The vesicles that are passively released in later phases of apoptosis are called apoptotic bodies. ...
... The cells turn from apoptosis to secondary necrosis [147]. The latter form of cell death mainly results, like primary necrosis, in immune activation and inflammatory reactions [63]. Uric acid has been identified as a danger signal released from dying cells [146]. ...
Lipids in the cytoplasm membrane fulfill numerous functions. We focus on how lipid asymmetry is generated and its physiological and pathophysiological mission. The role of phosphatidylserine (PS), a prominent phospholipid that gets exposed during cell death, in health and disease as well as in the clearance process will be outlined in detail. Attraction signals, bridging molecules, and danger signals being involved in the PS-dependent clearance of apoptotic and necrotic cells and in subsequent immune modulation are presented. Furthermore, modulations of immune responses by PS-exposing cells, organisms, microparticles, and by the PS-binding protein annexin A5 are discussed. Interference with PS-dependent clearance of apoptotic tumor cells by macrophages fosters uptake and presentation of cancer antigens by dendritic cells and thereby induces specific anti-tumor immunity. The lipid composition of microvesicles is also depicted. Tumor microvesicles are often rich in PS and thereby contribute to tumor escape mechanisms. Understanding the role of PS in membranes of dying cells and microvesicles will help to develop novel drugs and treatment options for controlling immune-mediated diseases like chronic autoimmunity and cancer.
... Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the development of autoreactivity against nuclear antigens, including double-stranded DNA (dsDNA) and histones [1,2,3]. The predominance of chromatin-associated antigen targets points at aberrancies in the processing and elimination of chromatin as a potential culprit of such a process [4,5,6,7,8]. It has been postulated that effective degradation of DNA from dying cells is essential to prevent priming of the immune system against chromatin self-antigens, and impaired chromatin degradation has been proposed as a mechanism for the development of antinuclear autoimmunity [9,10]. ...
... Heat inactivation of actin was achieved by incubation of samples in a heating block at 56uC for 10 minutes. Dnase1l3 inhibition was performed by addition of 4-([1,3,5]- triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396; Sigma-Aldrich) to a concentration of 5mM. Actin inhibition was achieved by pre-incubating the samples with 5mg/ mL bovine g-actin (Sigma-Aldrich) for 10 minutes. ...
Deposition of chromatin-IgG complexes within glomerular membranes is a key event in the pathogenesis of lupus nephritis. We recently reported an acquired loss of renal Dnase1 expression linked to transformation from mild to severe membranoproliferative lupus nephritis in (NZBxNZW)F1 mice. As this may represent a basic mechanism in the progression of lupus nephritis, several aspects of Dnase1 expression in lupus nephritis were analyzed.
Total nuclease activity and Dnase1 expression and activity was evaluated using in situ and in vitro analyses of kidneys and sera from (NZBxNZW)F1 mice of different ages, and from age-matched healthy controls. Immunofluorescence staining for Dnase1 was performed on kidney biopsies from (NZBxNZW)F1 mice as well as from human SLE patients and controls. Reduced serum Dnase1 activity was observed in both mesangial and end-stage lupus nephritis. A selective reduction in renal Dnase1 activity was seen in mice with massive deposition of chromatin-containing immune complexes in glomerular capillary walls. Mice with mild mesangial nephritis showed normal renal Dnase1 activity. Similar differences were seen when comparing human kidneys with severe and mild lupus nephritis. Dnase1 was diffusely expressed within the kidney in normal and mildly affected kidneys, whereas upon progression towards end-stage renal disease, Dnase1 was down-regulated in all renal compartments. This demonstrates that the changes associated with development of severe nephritis in the murine model are also relevant to human lupus nephritis.
Reduction in renal Dnase1 expression and activity is limited to mice and SLE patients with signs of membranoproliferative nephritis, and may be a critical event in the development of severe forms of lupus nephritis. Reduced Dnase1 activity reflects loss in the expression of the protein and not inhibition of enzyme activity.
... An impaired removal of apoptotic bodies also indirectly contributes to the pathogenesis of SLE. Histone-bound DNA complexes, which have high affinity for the glomerular basement membrane, are carried out from apoptotic cells and accumulated in the glomerulus, triggering an immune response and causing glomerular damage [148][149][150][151][152][153][154][155][156][157]. ...
... In den letzten Jahren wurde gezeigt, dass es auch eine programmierte Form der Nekrose gibt (Gaipl et al., 2006). Wichtig für die Auflösung von Tumoren oder Metastasen ist die Immunogenität der Tumorzellen, das bedeutet, es ist notwendig die therapieinduzierten Zelltodesarten dahingehend zu modulieren, dass das Immunsystem aktiviert wird und eine langanhaltende Anti-Tumor-Immunität entsteht. ...
Objectives Glioblastoma is the most common malignant neoplasia in the central nervous system in adults. Despite intensive research and therapy using multimodal treatment concepts, this brain tumor is still incurable and the average overall survival time of newly diagnosed patients is on average less than 15 months (Stupp et al., 2005). According to the current standard therapy, the tumor tissue is surgically resected as completely as possible and treated with adjuvant radi-ochemotherapy (RCT) consisting of temozolomide (TMZ) and fractionated irra-diation with 2 Gray (Gy) (Laperriere et al., 2002). The aim of radiochemotherapy is to induce cell death by apoptosis and necrosis by a proliferation stop in the tumor cells. In this thesis we investigated how the glioblastoma cell line U87 reacts to isolated radiotherapy as well as to combination therapies of radiation and TMZ in different doses. On the other hand, the glioblastoma cell line U87 was analyzed for necropto-sis. Recent investigations have shown that there are also other forms of cell death than necrosis and apoptosis, such as necroptosis. These cell death forms enable the development of alternative treatment strategies for resistant tumor cells. Necroptosis is immunogenic and, like apoptosis, a programmed form of cell death. In addition, this cell death form is receptor-mediated (recep-tor-activating protein kinase 1 (RIPK-1)), as is necrosis. Furthermore, as in ne-crosis, the cell loses its membrane integrity. In order to detect necroptosis, it can be inhibited with necrostatin (Nec-1) or triggered by blocking apoptosis (e.g. with z-VAD-fmk). Design & Methods In this thesis, the human glioblastoma cell line U87 was treated as a model system with chemotherapy consisting of TMZ and various Radiotherapies con-sisting of high single doses and fractionated irradiation with low single doses. Furthermore, these treatments were combined to RCT. In all samples, cell death and cell cycle determinations were performed by AxV / PI staining and flow cytometry and examined for apoptosis and necrosis. Irradiation was per-formed in single doses of 10 Gy, fractionated irradiation with 5 x 2 Gy and 2 x 5 Gy. The measurements were taken 24 h, 48 h and 72 h after the last treatment. TMZ was applied in a concentration of 20 μM. In addition, the influence of RCT on necroptosis induction was investigated. On the one hand, necroptosis was inhibited by Nec-1, which could be detected by reducing the necrotic cell fraction compared to the sample without Nec-1. On the other hand, the induction of necroptosis by inhibition of apoptosis us-ing z-VAD-fmk was investigated, which would be indicated by a reduced pro-portion of apoptotic cells compared to samples without z-VAD-fmk. Observations & Results In this study it could be shown that the glioblastoma cell line U87 reacts signif-icantly to high single doses (10 Gy) after 48 h and 72 h, both with apoptosis and with necrosis. Furthermore, it could be shown that a fractionation of the irradiation with 2 x 5 Gy leads to a further significant increase of the apoptotic and necrotic cells from approx. 3 % after 48 h to approx. 10 % necrotic and ap-prox. 5 % apoptotic cells. The combination of radio and chemotherapy is simi-lar. After 72 h and 48 h and treatment with 10 Gy and TMZ, a significant in-crease in cell mortality was achieved. Even more effective than the combina-tion of single doses and TMZ is the combination of fractionated irradiation and TMZ, because the proportion of necrotic and apoptotic cells increases after 48 h from about 4 % to about 14 % necrotic and about 10 % apoptotic cells. The examination of the cell line U87 for necroptosis was performed by inhibi-tion by Nec-1 and induction by z-VAD-fmk. Nec-1 was not shown to significant-ly reduce the proportion of necroptotic cells. Only with a therapy combination of 10 Gy, TMZ and z-VAD-fmk after 48 h and 72 h a reduction of the proportion of apoptotic cells could be achieved, whereas at the same time there was only an increase of necrotic cells with a treatment with 10 Gy and Nec-1. This suggests that glioblastoma cells of cell line U87 are not or not significantly capable of necroptosis. Conclusions It could be shown in this thesis that cell mortality by radiotherapy with fraction-ated irradiation is more effective than with high single doses. Furthermore, the combination of fractionated irradiation and TMZ with RCT with TMZ and high single doses led to a significant increase in apoptosis and necrosis. A combi-nation of 2 x 5 Gy and TMZ was able to achieve the best effect. Furthermore, the results of the necroptosis analysis indicate that the glioblas-toma cells of cell line U87 are not or not significantly capable of necroptosis. However, it remains to be investigated whether after these treatments there are danger signals in the supernatant cells which nevertheless increase the im-munogenicity.
... Cancer cells represent abnormal cells and express aberrant antigens in microenvironment that can promote antigen generation [19,20]. The immune system appears to be sensitive to aberrant structures, distributions, and functions of neoplastic components and incites immune responses to TAAs, generating TAAbs [21][22][23][24]. The mechanisms of TAAbs production in cancer are summarized as follows: (1) defects in tolerance and inflammation, (2) overexpression of TAA or TAA expression in aberrant location, (3) altered protein structure caused by neoepitopes, mutations, or post-translational modifications, and (4) cellular death mechanisms [25] (Fig. 2). ...
Lung cancer (LC) accounts for the majority of cancer-related deaths worldwide. Although screening the high-risk population by low-dose CT (LDCT) has reduced mortality, the cost and high false positivity rate has prevented its general diagnostic use. As such, better and more specific minimally invasive biomarkers are needed in general and for early LC detection, specifically. Autoantibodies produced by humoral immune response to tumor-associated antigens (TAA) are emerging as a promising noninvasive biomarker for LC. Given the low sensitivity of any one single autoantibody, a panel approach could provide a more robust and promising strategy to detect early stage LC. In this review, we summarize the background of TAA autoantibodies (TAAb) and the techniques currently used for identifying TAA, as well as recent findings of LC specific antigens and TAAb. This review provides guidance toward the development of accurate and reliable TAAb as immunodiagnostic biomarkers in the early detection of LC.
... Apoptotic eosinophils are known to be eliminated by professional phagocytes, including resident or recruited monocytes-macrophages as well as by "non-professional" phagocytes such as epithelial cells via efferocytosis (Fig. 2), a crucial process for the resolution of the airway inflammation [12,13]. In fact, defects in apoptosis and/or subsequent efferocytosis of inflammatory cells are known to be involved in chronic inflammatory diseases of the lung and autoimmune diseases [7,14]. Several agents are known to prolong eosinophil survival by delaying apoptosis, including the cytokines GM-CSF, IL-3, and IL-5 [3,15,16]. ...
It is becoming increasingly clear that nanoparticles (NPs) possess many potential applications in both clinical medicine and research. Potential utilization of NPs in nanomedicine for the treatment of respiratory diseases where eosinophils exert pathogenic roles is gaining increasing attention. Even though several NPs were found to possess pro-inflammatory activities in in vivo models based on an increased number of eosinophils in rodent airways, it is not clear how NPs could directly activate eosinophils themselves and how they can alter their biology. In this review, we discuss the most recent data in this new area of research demonstrating that NPs could now be added as new eosinophils modulators. Indeed, activation of eosinophils with NPs could lead to modulation of spontaneous apoptosis, caspase activation, and cytoskeleton breakdown when apoptosis is induced; cytokine production, de novo protein synthesis, cellular adhesion onto a cell substratum, and cell signalling events such as activation of the phosphoinositide 3-kinase/Akt pathway and actin re-localization are involved in NP-induced adhesion. Therefore, future development of therapeutic strategies with NPs aiming at targeting diseases where eosinophils are involved should now consider the capacity of NPs to modulate human eosinophil biology.
... An impaired removal of apoptotic bodies also indirectly contributes to the pathogenesis of SLE. Histone-bound DNA complexes, which have high affinity for the glomerular basement membrane, are carried out from apoptotic cells and accumulated in the glomerulus, triggering an immune response and causing glomerular damage [120][121][122][123][124][125][126][127][128][129]. ...
Apoptotic cell death is usually a response to the cell’s microenvironment. In the kidney, apoptosis contributes to parenchymal cell loss in the course of acute and chronic renal injury, but does not trigger an inflammatory response. What distinguishes necrosis from apoptosis is the rupture of the plasma membrane, so necrotic cell death is accompanied by the release of unprocessed intracellular content, including cellular organelles, which are highly immunogenic proteins. The relative contribution of apoptosis and necrosis to injury varies, depending on the severity of the insult. Regulated cell death may result from immunologically silent apoptosis or from immunogenic necrosis. Recent advances have enhanced the most revolutionary concept of regulated necrosis. Several modalities of regulated necrosis have been described, such as necroptosis, ferroptosis, pyroptosis, and mitochondrial permeability transition-dependent regulated necrosis. We review the different modalities of apoptosis, necrosis, and regulated necrosis in kidney injury, focusing particularly on evidence implicating cell death in ectopic renal calcification. We also review the evidence for the role of cell death in kidney injury, which may pave the way for new therapeutic opportunities.
... If exposed chromatin is potentially dangerous to the body, as discussed by e.g., Darrah and Andrade (24) and by others (25)(26)(27)(28)(29), this may illuminate how important it is to remove chromatin from dying cells in an abrupt and silent way. However, in individuals with anti-dsDNA antibodies and impaired clearance of cell debris including necrotic chromatin, like in SLE (30)(31)(32)(33)(34)(35)(36)(37), this may change the situation from a controlled removal of chromatin into a condition where chromatin debris remains exposed and may be a contributor to produce and or amplify anti-dsDNA antibodies by interaction with TLR9 and to promote inflammation. This may be caused by slow removal of extra-cellular dsDNA by e.g., silencing of DNase I or blocking of DNase I activity since binding of anti-dsDNA antibodies to DNA may inhibit the effect of the endonucleases [discussed in (29)]. ...
This study aims to understand what lupus nephritis is, its origin, clinical context, and its pathogenesis. Truly, we encounter many conceptual and immanent tribulations in our attempts to search for the pathogenesis of this disease—and how to explain its assumed link to SLE. Central in the present landscape stay a short history of the early studies that substantiated the structures of isolated or chromatin-assembled mammalian dsDNA, and its assumed, highly controversial role in induction of anti-dsDNA antibodies. Arguments discussed here may provoke the view that anti-dsDNA antibodies are not what we think they are, as they may be antibodies operational in quite different biological contexts, although they bind dsDNA by chance. This may not mean that these antibodies are not pathogenic but they do not inform how they are so. This theoretical study centers the content around the origin and impact of extra-cellular DNA, and if dsDNA has an effect on the adaptive immune system. The pathogenic potential of chromatin-anti-dsDNA antibody interactions is limited to incite lupus nephritis and dermatitis which may be linked in a common pathogenic process. These are major criteria in SLE classification systems but are not shared with other defined manifestations in SLE, which may mean that they are their own disease entities, and not integrated in SLE. Today, the models thought to explain lupus nephritis are divergent and inconsistent. We miss a comprehensive perspective to try the different models against each other. To do this, we need to take all elements of the syndrome SLE into account. This can only be achieved by concentrating on the interactions between autoimmunity, immunopathology, deviant cell death and necrotic chromatin in context of elements of system science. System science provides a framework where data generated by experts can be compared, and tested against each other. This approach open for consensus on central elements making up “lupus nephritis” to separate what we agree on and how to understand the basis for conflicting models. This has not been done yet in a systematic context.
... Additionally, activation of mTOR was shown in SLE patients which further suggests an association between SLE and the autophagy machinery (Pierdominici et al., 2012). In the context of autophagydeficiency, it was discussed that one of the potential causes of SLE might be the defective clearance of apoptotic cell corpses which might lead to the breakdown of self-tolerance, the activation of autoreactive lymphocytes and, finally, to the development of SLE (Gaipl et al., 2006). ...
Zusammenfassung Multiple Sklerose (MS) ist eine Autoimmunerkrankung des zentralen Nervensystems (ZNS). Untersuchungen des Mausmodells für MS, namentlich Experimentelle autoimmune Enzephalomyelitis (EAE), konnten zeigen, dass autoreaktive CD4+ T-Zellen eine herausragende Rolle in der Entwicklung von EAE spielen. Jedoch ist bisher unklar, ob Selbstantigene des Myelins über den klassischen MHC Klasse II Prozessierungsweg, Autophagie oder LC3-assoziierte Phagozytose (LAP) degradiert und gegenüber CD4+ T-Zellen präsentiert werden. Mit Bezug auf LAP konnte gezeigt werden, dass extrazelluläres Material über diesen Weg abgebaut werden kann und zudem nimmt die Anzahl von LC3+ Vesikeln in Microglia-Zellen am Höhepunkt der EAE zu. Basierend auf diesen Daten haben wir angenommen, dass die Präsentation von Autoantigenen durch LAP die Aktivierung von autoreaktiven T-Zellen unterstützt. Um diese Hypothese zu bestätigen, haben wir den Einfluss von Atg5-Defizienz in CD11c+ Zellen auf die Entwicklung der EAE untersucht. Wir haben gezeigt, dass die Defizienz von Atg5 in CD11c+ Zellen keine Rolle bei der Entwicklung von aktiver EAE spielt und dass dieses Ergebnis davon unabhängig ist, ob MOG-Peptid oder MOG-Protein zur Induktion der Krankheit verwendet worden ist. Überraschenderweise resultierte die Induktion der adoptiven Transfer-EAE darin, dass EAE- Symptome in Atg5-defizienten Mäusen nahezu abwesend waren, während Atg5-Kontroll Mäuse normale EAE-Symptome entwickelt haben. Um diese gegensätzlichen Ergebnisse besser verstehen zu können, wurden MOG-spezifische T-Zellen mit primären Atg5+ oder Atg5- CD11c+ Zellen, in der Gegenwart von MOG-Peptid, MOG-Protein oder Kugeln, welche mit MOG-Protein beschichtet waren, co-kultiviert. Hierbei ergab die Messung der Proliferation von T-Zellen, dass T Zellen in der Gegenwart von Atg5- CD11c+ Zellen mehr proliferieren als in der Gegenwart von Atg5+ CD11c+ Zellen. Um die Antigenpräsentation weitergehend zu untersuchen, wurden sowohl Atg5+ als auch Atg5- BM-DCs generiert, die mit Kugeln inkubiert wurden, welche MOG-Protein, Pam3CSK4 oder MOG-Protein plus Pam3CSK4 beschichtet waren. Bei diesen Untersuchungen konnten keine LC3-assoziierten Phagosomen erkannt werden und somit keine Rückschlüsse über die Rolle von LAP während der Prozessierung von extrazellulären Autoantigenen gezogen werden. Weitere in vitro und in vivo Experimente werden notwendig sein, um die unterschiedlichen Ergebnisse in der aktiven und adoptiven Transfer-EAE erklären zu können. Zum einen müssten die T-Zell- und Antigenpräsentierende Zellinfiltrate in das ZNS am Höhepunkt der Krankheit untersucht werden. Diese Untersuchungen könnten darüber Aufschluss geben, welche Zellen das ZNS infiltrieren und welche Effektorfunktionen diese besitzen. Darüber hinaus könnte der Antigenprozessierungsweg untersucht werden, indem LAP durch bekannte Faktoren, wie beispielsweise TLR-Stimulierung, FcR-Bindung oder mit der Verwendung von “eat me” Signalen, ausgelöst wird. Alle Experimente zusammengenommen, könnten somit neue Einblicke dahin gehend ermöglichen, über welchen Prozessierungsweg extrazelluläre Autoantigene verarbeitet werden und wie CD4+ autoreaktive T-Zellen dazu aktiviert werden, EAE oder MS zu induzieren. Summary Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). The commonly used mouse model for MS, namely experimental autoimmune encephalomyelitis (EAE), revealed that the main driving forces in the development of EAE are autoreactive CD4+ T cells. The exact antigen processing route of how myelin self-antigens are processed and presented has not been described yet and the classical MHC class II pathway, the autophagy pathway and LC3- associated phagocytosis (LAP) are potential degradation pathways that might lead to MHC class II presentation of myelin antigen. It has recently been shown that extracellular material can be degraded by LAP and previous studies of our laboratory indicate that the presence of LC3+ vesicles in microglia increases at the peak of EAE. Based on these data, we hypothesized that LAP-dependent presentation of autoantigens facilitates the activation of autoreactive T cells. To address this question, we investigated whether autophagy deficiency in CD11c+ cells influences the development of EAE. We demonstrated that Atg5-deficiency in CD11c+ cells does not play a role in active EAE, neither triggered by MOG peptide nor MOG protein. Surprisingly, induction of adoptive transfer EAE via the adoptive transfer of autoreactive CD4+ T cells resulted in the almost complete absence of EAE symptoms in Atg5-deficient mice whereas Atg5 control mice developed EAE symptoms. In order to better understand these findings, in vitro experiments to study the role of Atg5 in antigen presentation were performed. A co-culture experiment in which MOG peptide-specific T cell receptor transgenic T cells from 2D2 mice were co-cultured with splenic Atg5+ or Atg5- CD11c+ cells in the presence of MOG peptide, MOG protein or beads coated with MOG protein was performed. Checking for T cell proliferation, it was shown that T cells in the presence of Atg5- CD11c+ proliferated more than in the presence of Atg5+ CD11c+ cells. In an alternative approach to investigate antigen processing pathways in antigen presenting cells (APCs), bone marrow-derived dendritic cells (BM-DCs) were generated from Atg5-deficient and Atg5 control mice and incubated with beads coated with MOG protein, the TLR agonist Pam3CSK4 or MOG protein with Pam3CSK4. Screening for LC3+ vesicles that might be an indication for LAP, did not yield any unambigous LC3- associated phagosomes. Conclusively, further in vitro and in vivo investigations are necessary to identify the mechanism that leads to the absence of EAE symptoms in Atg5-deficient mice in adoptive transfer EAE, but not in active EAE. Firstly, T cell and APC infiltrates into the CNS at peak of disease need to be examined. These experiments would offer valuable clues which cells infiltrate and which effector functions they fulfill. With regard to in vitro studies, antigen processing and presentation characteristics could be studied by inducing LAP by known factors such as TLR stimulation, Fc receptor (FcR) engagement or “eat me” signals. In conclusions, our findings would give newly insights of how extracellular self-antigen is processed and how CD4+ autoreactive T cells are triggered to induce EAE, or even MS.
... Furthermore, with regard to radiation protection issues, the maintained functional competence of macrophages after exposure to irradiation is reassuring, as invading pathogens or damaged cells can still be cleared properly. To avoid autoimmune reactions in the body, the latter is of great importance [42]. Before monocytes/macrophages can properly clear infected, damaged and/or dying cells, they have to find the target [43]. ...
... As an initially unanticipated result of the pilot experiments, the morphology of GC Mφ and associated apoptotic cells of NZB/W mice turned out to be obviously peculiar. Because the generation of high titers of anti-DNA antibodies in autoimmune mice is generally ascribed to malfunction of GC and in particular to clearing defects of Mφ within GC [150], the NZB/W model emerged as an eligible candidate to complement the analysis of the kinetics of apoptotic cells and their uptake by Mφ assessed in BALB/c mice. Therefore, the follicular Mφ of NZB/W mice were subjected to further analysis. ...
Ein zentrales Merkmal der humoralen Antwort ist die im Laufe der Zeit ansteigende Affinität der Antikörper gegenüber dem Antigen, ein Phänomen, das man generell als Affinitätsreifung bezeichnet. Die Affinitätsreifung von Antikörpern ist an die transiente Ausbildung von Keimzentren gebunden, die man nach Immunisierung mit einem T-Zell-abhängigen Antigen in sekundär lymphatischen Geweben wie der Milz beobachtet. Innerhalb der Keimzentren durchlaufen B-Zellen einen mikro-evolutionären Prozess, in dessen Verlauf es zu einer Diversifizierung der von den B-Zellen kodierten B-Zell-Rezeptoren durch somatische Hypermutation und anschließender Selektion derjenigen B-Zellen mit den besten Bindungseigenschaften gegenüber dem Antigen kommt. In den letzten Jahren waren große Fortschritte hinsichtlich der Aufklärung der molekularen Mechanismen die zur Diversifizierung der B-Zell-Rezeptoren beitragen zu verzeichnen, wohingegen die Dynamik, der Mechanismus und die treibenden Kräfte der Selektion bisher weitgehend unverstanden sind. In Kürze zusammengefasst trägt die vorliegende Arbeit durch folgende Erkenntnisse zum Verständnis der Evolution von B-Zellen in Keimzentren bei: (1) nicht-synchronisiertes Wachstumsverhalten von Keimzentren, (2) mehrstufige Selektionsstrategie (Verknüpfung von lokaler und globaler Selektion durch Rezirkulation von ausgewanderten Keimzentrums-B-Zellen), (3) zentrale Rolle von Makrophagen für die Homöostase von Keimzentren und die Verhinderung von Autoimmunität, (4) bisher angenommene molekulare Signaturen sind unzulänglich, um positiv und negativ selektierte B-Zellen zu unterscheiden und (5) das Überleben bzw. positive Selektion von Keimzentrums-B-Zellen ist von der Abwesenheit überschüssiger, nachteiliger Mutationen in den CDRs abhängig.
... They show particularly high affinity for nucleic acid (dsDNA) as well as for nuclear and cytoplasmic proteins that physically interact with nucleic acids, although individual reactivity patterns are highly heterogeneous. Autoantibody generation, in part originating from inappropriately processed self-antigens [1], is clearly T cell-dependent and occurs during germinal center reactions leading to a high degree of somatic hypermutation [2]. In these reactions, T-and B-cell targets can diversify by intra-and intermolecular epitope spreading, a mechanism in which crossreactivity and aberrant interactions between specific T-and B-cells favor the selection of self-reactive B-cells. ...
... Damage-associated molecular patterns (DAMPs) are released because the plasma membrane of necrotic cells is disturbed. 7,8 Danger signals as the high mobility group protein B1 (HMGB1) and the nucleotide adenosine triphosphate (ATP) activate DCs, foster crosspresentation of antigens and consecutively the activation of T cells. 9 DAMPs therefore link radio-and/or chemotherapyinduced local alterations of the tumor cells and subsequent systemic anti-tumor immune reactions. ...
One prerequisite that radiotherapy (RT) and chemotherapy (CT) result in anti-tumor immune responses is triggering of immunogenic cell death forms such as necroptosis. The latter is inducible by inhibition of apoptosis with the pan-caspase inhibitor zVAD-fmk. The design of multimodal therapies that overcome melanoma's resistance to apoptosis is a big challenge of oncoimmunology. As hints exist that immune stimulation by hyperthermia (HT) augments the efficacy of melanoma therapies and that tumors can be sensitized for RT with zVAD-fmk, we asked whether combinations of RT with dacarbazine (DTIC) and/or HT induce immunogenic melanoma cell death and how this is especially influenced by zVAD-fmk. Necroptosis was inducible in poorly immunogenic B16-F10 melanoma cells and zVAD-fmk generally increased melanoma cell necrosis concomitantly with the release of HMGB1. Supernatants (SNs) of melanoma cells whose cell death was modulated with zVAD-fmk induced an upregulation of the activation markers CD86 and MHCII on macrophages. The same was seen on dendritic cells (DCs), but only when zVAD-fmk was added to multimodal tumor treatments including DTIC. DCs of MyD88 KO mice and DCs incubated with SNs containing apyrase did not increase the expression of these activation markers on their surface. The in vivo experiments revealed that zVAD-fmk decreases the tumor growth significantly and results in a significantly reduced tumor infiltration of Tregs when added to multimodal treatment of the tumor with RT, DTIC and HT. Further, a significantly increased DC and CD8+ T-cell infiltration into the tumor and in the draining lymph nodes was induced, as well as an increased expression of IFNγ by CD8+ T cells. However, zVAD-fmk did not further reduce tumor growth in MyD88 KO mice, mice treated with apyrase or RAG KO mice. We conclude that HMGB1, nucleotides and CD8+ T cells mediate zVAD-fmk induced anti-melanoma immune reactions in multimodal therapy settings.
... 23 Circulating DNA from dying cells is rapidly degraded by DNAse. 24 The large titers of plasma DNA in trauma patients suggests deviation from normal physiologic process. ...
Mitochondrial DNA (mtDNA), a potent proinflammatory damage-associated molecular pattern, is released in large titers following trauma. The effect of trauma surgery on mtDNA concentration is unknown. We hypothesized that mtDNA and nuclear DNA (nDNA) levels would increase proportionately with the magnitude of surgery and both would then decrease rapidly.
In this prospective pilot, plasma was sampled from 35 trauma patients requiring orthopedic surgical intervention at six perioperative time points. Healthy control subjects (n = 20) were sampled. DNA was extracted, and the mtDNA and nDNA were assessed using quantitative polymerase chain reaction. Markers of cell necrosis were also assayed (creatine kinase, lactate dehydrogenase, and aspartate aminotransferase).
The free plasma mtDNA and nDNA levels (ng/mL) were increased in trauma patients compared with healthy controls at all time points (mtDNA: preoperative period, 108 [46-284]; postoperative period, 96 [29-200]; 7 hours postoperatively, 88 [43-178]; 24 hours, 79 [36-172]; 3 days, 136 [65-263]; 5 days, 166 [101-434] [healthy controls, 11 (5-19)]) (nDNA: preoperative period, 52 [25-130]; postoperative period, 100 [35-208]; 7 hours postoperatively, 75 [36-139]; 24 hours postoperatively, 85 [47-133]; 3 days, 79 [48-117]; 5 days, 99 [41-154] [healthy controls, 29 (16-54)]). Elevated DNA levels did not correlate with markers of cellular necrosis. mtDNA was significantly elevated compared with nDNA at preoperative period (p = 0.003), 3 days (p = 0.003), and 5 days (p = 0.0014). Preoperative mtDNA levels were greater with shorter time from injury to surgery (p = 0.0085). Postoperative mtDNA level negatively correlated with intraoperative crystalloid infusion (p = 0.0017). Major pelvic surgery (vs. minor) was associated with greater mtDNA release 5 days postoperatively (p < 0.05).
This pilot of heterogeneous orthopedic trauma patients showed that the release of mtDNA and nDNA is sustained for 5 days following orthopedic trauma surgery. Postoperative, circulating DNA is not associated with markers of tissue necrosis but is associated with surgical invasiveness and is inversely related to intraoperative fluid administration. Sustained elevation of mtDNA levels could be of inflammatory origin and may contribute to postinjury dysfunctional inflammation.
Prospective study, level III.This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License, where it is permissible to download and share thework provided it is properly cited. Thework cannot be changed in any way or used commercially.
... Failure or inhibition of these clearance mechanisms permits the apoptotic cells to enter into secondary necrosis, which results in injurious perpetuation of the inflammatory response. 52 Indeed, defects in apoptosis and/or subsequent efferocytosis of pro-inflammatory cells are increasingly recognized in conditions as diverse as autoimmunity 53 and chronic inflammatory diseases of the lung. 54 Evidence for the rationale of targeting apoptosis/ efferocytosis in respiratory disease is provided by studies that demonstrated defects in recognition of apoptotic cells by alveolar macrophages from patients with COPD 55 or chronic asthma. ...
Asthma is an increasingly common respiratory condition characterized by reversible airway obstruction, bronchial hyper-responsiveness and airway inflammation with a clear unmet need for more effective therapy. Eosinophilic asthma is a phenotype of the condition that features increased blood or sputum eosinophils whose numbers correlate with disease severity. Several lines of evidence are now emerging, which implicate increased persistence of eosinophils in the lungs of patients with asthma as a consequence of inhibition of and defects in the apoptotic process, together with impaired apoptotic cell removal mechanisms. This article will update our knowledge of the mechanisms controlling eosinophil apoptosis and clearance, together with evidence implicating defects in apoptosis and pro-inflammatory cell removal in asthma. Recent developments in novel therapies for asthma that target eosinophil apoptotic and/or clearance pathways will also be discussed.
... Furthermore, with regard to radiation protection issues, the maintained functional competence of macrophages after exposure to irradiation is reassuring, as invading pathogens or damaged cells can still be cleared properly. To avoid autoimmune reactions in the body, the latter is of great importance [42]. Before monocytes/macrophages can properly clear infected, damaged and/or dying cells, they have to find the target [43]. ...
Benign painful and inflammatory diseases are treated for decades with low/moderate doses of ionizing radiation (LD-X-irradiation). Tissue macrophages regulate initiation and resolution of inflammation by the secretion of cytokines and by acting as professional phagocytes. Having these pivotal functions, we were interested in how activated macrophages are modulated by LD-X-irradiation, also with regard to radiation protections issues and carcinogenesis.We set up an ex-vivo model in which lipopolysaccharide pre-activated peritoneal macrophages (pMΦ) of radiosensitive BALB/c mice, mimicking activated macrophages under inflammatory conditions, were exposed to X-irradiation from 0.01 Gy up to 2 Gy. Afterwards, the viability of the pMΦ, their transmigration and chemotaxis, the phagocytic behavior, the secretion of inflammatory cytokines and underlying signaling pathways were determined.Exposure of pMΦ up to a single dose of 2 Gy did not influence their viability and phagocytic function, an important fact regarding radiation protection. However, a significantly reduced migration, but an increased chemotaxis of pMΦ after exposure to 0.1 or 0.5 Gy was detected. Both might get along with resolution of inflammation. Cytokine analyses revealed that especially the moderate dose of 0.5 Gy applied in low dose radiotherapy for inflammatory diseases results in an anti-inflammatory cytokine microenvironment of pMΦ, as the secretion of the pro-inflammatory cytokine IL-1ß was reduced and that of the anti-inflammatory cytokine TGF-ß increased. Further, the reduced secretion of IL-1ß correlated with reduced nuclear translocation of NFкB p65, starting at exposure of pMΦ to 0.5 Gy of X-irradiation.We conclude that inflammation is modulated by LD-X-irradiation via changing the inflammatory phenotype of macrophages.
... The latter is inhibitable by necrostatin-1, while caspase-dependent apoptosis is inhibitable by the pancaspase inhibitor zVAD-fmk [32,33]. Secondary necrotic cells result from apoptotic cells that were not properly cleared and have lost their membrane integrity at any time point during apoptosis execution [34,35]. Primary nonprogrammed necrosis is characterized by early plasma membrane rupture and dilatation of cytoplasmic organelles [36]. ...
Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Despite a multimodal therapy consisting of resection followed by fractionated radiotherapy (RT) combined with the chemotherapeutic agent (CT) temozolomide (TMZ), its recurrence is almost inevitable. Since the immune system is capable of eliminating small tumor masses, a therapy should also aim to stimulate anti-tumor immune responses by induction of immunogenic cell death forms. The histone deacetylase inhibitor valproic acid (VPA) might foster this.
Reflecting therapy standards, we applied in our in vitro model fractionated RT with a single dose of 2Gy and clinically relevant concentrations of CT. Not only the impact of RT and/or CT with TMZ and/or VPA on the clonogenic potential and cell cycle of the glioblastoma cell lines T98G, U251MG, and U87MG was analyzed, but also the resulting cell death forms and release of danger signals such as heat-shock protein70 (Hsp70) and high-mobility group protein B1 (HMGB1).
The clonogenic assays revealed that T98G and U251MG, having mutated tumor suppressor protein p53, are more resistant to RT and CT than U87MG with wild type (WT) p53. In all glioblastoma cells lines, fractionated RT induced a G2 cell cycle arrest, but only in the case of U87MG, TMZ and/or VPA alone resulted in this cell cycle block. Further, fractionated RT significantly increased the number of apoptotic and necrotic tumor cells in all three cell lines. However, only in U87MG, the treatment with TMZ and/or VPA alone, or in combination with fractionated RT, induced significantly more cell death compared to untreated or irradiated controls. While necrotic glioblastoma cells were present after VPA, TMZ especially led to significantly increased amounts of U87MG cells in the radiosensitive G2 cell cycle phase. While CT did not impact on the release of Hsp70, fractionated RT resulted in significantly increased extracellular concentrations of Hsp70 in p53 mutated and WT glioblastoma cells.
Our results indicate that fractionated RT is the main stimulus for induction of glioblastoma cell death forms with immunogenic potential. The generated tumor cell microenvironment might be beneficial to include immune therapies for GBM in the future.
... For example, engagement of TLRs can upregulate the expression of proinflammatory cytokines (tumor necrosis factor [TNF]α and interferon [IFN]γ [71]) and interleukins (72,73) that directly induce expression of MMPs (71)(72)(73). Alternatively, incomplete clearance and degradation of apoptotic cells may transform them into secondary necrotic cell debris (63,74). This step may be significant, since necrotic cell debris contains SAP130, which serves as a ligand for the inflammation-related receptor Clec4e (75,76). ...
Autoantibodies to components of chromatin, which includes dsDNA, histones, and nucleosomes are regarded central in the pathogenesis of lupus nephritis. How anti-chromatin autoantibodies exert their nephritogenic activity is, however, controversial. One model assumes that autoantibodies initiate inflammation when they cross-react with intrinsic glomerular structures like components of membranes, matrices or with exposed non-chromatin ligands released from cells. Another model suggests glomerular deposition of autoantibodies in complex with chromatin, thereby inducing a classical immune complex mediated tissue damage. Recent data suggest an acquired error of renal chromatin degradation due to the loss of renal DNaseI enzyme activity to be an important contributing factor for the development of lupus nephritis in lupus-prone (NZBxNZW)F1 mice and in patients with lupus nephritis. Down-regulation of DNaseI expression results in reduced chromatin fragmentation, and in deposition of extracellular chromatin-IgG complexes in glomerular basement membranes in individuals that produce IgG anti-chromatin autoantibodies. The main focus of the present review is to discuss whether exposed chromatin fragments in glomeruli are targeted by potentially nephritogenic anti-dsDNA autoantibodies, or if the nephritogenic activity of these autoantibodies is explained by cross-reaction with intrinsic glomerular constituents, or if both models co-exist in diseased kidneys. In addition the role of silencing of the renal DNaseI gene and the biological consequences of reduced chromatin fragmentation in nephritic kidneys are discussed.
... Some cell death forms are highly genetically determined ; others display more accidental characteristics (Griffith and Ferguson, 2011). Most attention was given to the two main cell death forms, namely apoptosis and necrosis, since their immune modulatory potential helped to understand how chronic autoimmune diseases might develop and/or sustained (Gaipl et al., 2006; Gaipl, 2009 ). Usually, apoptotic cells are immunologically silent or even tolerogenic. ...
Multimodal approaches are nowadays successfully applied in cancer therapy. Primary locally acting therapies such as radiotherapy (RT) and surgery are combined with systemic administration of chemotherapeutics. Nevertheless, the therapy of cancer is still a big challenge in medicine. The treatments often fail to induce long lasting anti-tumor responses. Tumor recurrences and metastases result. Immunotherapies are therefore ideal adjuncts to standard tumor therapies since they aim to activate the patient`s immune system against malignant cells even outside the primary treatment areas (abscopal effects). Especially cancer vaccines may have the potential both to train the immune system against cancer cells and to generate an immunological memory, resulting in long-lasting anti-tumor effects. However, despite promising results in phase I and II studies, most of the concepts finally failed. There are some critical aspects in development and application of cancer vaccines that may decide on their efficiency. The time point and frequency of medication, usage of an adequate immune adjuvant, the vaccine’s immunogenic potential, and the tumor burden of the patient are crucial. Whole tumor cell vaccines have advantages compared to peptide-based ones since a variety of tumor antigens are present. The master requirements of cell-based, therapeutic tumor vaccines are the complete inactivation of the tumor cells and the increase of their immunogenicity. Since the latter is highly connected with the cell death modality, the inactivation procedure of the tumor cell material may significantly influence the vaccine’s efficiency. We therefore also introduce high hydrostatic pressure (HHP) as an innovative inactivation technology for tumor cell based vaccines and outline that HHP efficiently inactivates tumor cells by enhancing their immunogenicity. Finally studies are presented proving that anti-tumor immune responses can be triggered by combining RT with selected immune therapies.
... It has been hypothesized, that under certain circumstances these intracellular autoantigens become visible to the immune system when they accumulate during apoptosis. In fact the impaired clearance of apoptotic cell debris and dsDNA by macrophages might induce TLR signaling and differentiation of autoreactive B cells (Gaipl et al., 2006). Response to nucleic acid-containing immunecomplexes relies on the coengagement of endosomal members of the TLR family, TLR9 and TLR7 (Marshak-Rothstein, 2006). ...
Genetic defects in the adenosine deaminase (ADA) gene are among the most common causes for severe combined immunodeficiency (SCID). ADA-SCID patients suffer from lymphopenia, severely impaired cellular and humoral immunity, failure to thrive, and recurrent infections. Currently available therapeutic options for this otherwise fatal disorder include bone marrow transplantation (BMT), enzyme replacement therapy with bovine ADA (PEG-ADA), or hematopoietic stem cell gene therapy (HSC-GT). Although varying degrees of immune reconstitution can be achieved by these treatments, breakdown of tolerance is a major concern in ADA-SCID. Immune dysregulation such as autoimmune hypothyroidism, diabetes mellitus, hemolytic anemia, and immune thrombocytopenia are frequently observed in milder forms of the disease. However, several reports document similar complications also in patients on long-term PEG-ADA and after BMT or GT treatment. A skewed repertoire and decreased immune functions have been implicated in autoimmunity observed in certain B-cell and/or T-cell immunodeficiencies, but it remains unclear to what extent specific mechanisms of tolerance are affected in ADA deficiency. Herein we provide an overview about ADA-SCID and the autoimmune manifestations reported in these patients before and after treatment. We also assess the value of the ADA-deficient mouse model as a useful tool to study both immune and metabolic disease mechanisms. With focus on regulatory T- and B-cells we discuss the lymphocyte subpopulations particularly prone to contribute to the loss of self-tolerance and onset of autoimmunity in ADA deficiency. Moreover we address which aspects of immune dysregulation are specifically related to alterations in purine metabolism caused by the lack of ADA and the subsequent accumulation of metabolites with immunomodulatory properties.
... However, apoptotic tumor cells may use the immune suppressive environment which is induced by PS as a strategy for tumor escape [96]. A failure in the clearance of apoptotic tumor cells can initiate secondary necrosis which originates from the apoptotic cells that have lost their membrane integrity during the course of programmed cell death [97]. In contrast to apoptosis, necrosis promotes inflammatory responses and in general, the immunogenic form of tumor cell death induced by necrosis dominates over that of apoptosis [98]. ...
Although surgery and radiotherapy are highly efficient in local tumor control, distal metastases and tumor recurrence often limit therapeutic outcome. It is becoming progressively more evident that curative tumor therapy depends on the presence and maintenance of an intact immune system which has the capacity to elicit cytotoxic effector functions against circulating tumor cells and distant metastases. Heat shock proteins (HSPs, also termed stress proteins) are involved in antigen processing and presentation and can act as "danger signals" for the adaptive and innate immune systems. This article reviews current knowledge relating to the induction and manifestation of stress protein-related immunological responses that are pertinent to the development and maintenance of protective anti-tumor immunity.
... However, in chronic autoimmune diseases the clearance machinery of the body is overcharged and apoptotic cells accumulate. The latter lose their membrane integrity during time and are then called secondary necrotic cells (Gaipl et al., 2006). A disturbed clearance of the body's own cells therefore might lead to chronic autoimmune disease, as has been described by our group and by others with respect to systemic lupus erythematosus (Botto, 2001; Baumann et al., 2002). ...
Chemotherapeutic agents (CT) and ionizing radiation (X-ray) induce DNA damage and primarily aim to stop the proliferation of tumor cells. However, multimodal anti-cancer therapies should finally result in tumor cell death and, best, in the induction of systemic anti-tumor immunity. Since distinct therapy-induced tumor cell death forms may create an immune activating tumor microenvironment, this study examined whether sole treatment with CT that are used in the therapy for colorectal cancer or in combination with X-ray result in colorectal tumor cell death with immunogenic potential. 5-Fluorouracil (5-FU), Oxaliplatin (Oxp), or Irinotecan (Irino) in combination with X-ray were all potent inhibitors of colorectal tumor cell colony formation. This study then examined the forms of cell death with AnnexinA5-FITC/Propidium Iodide staining. Necrosis was the prominent form of cell death induced by CT and/or X-ray. While only a combination of Irino with X-ray leads to death induction already 1 day after treatment, also the combinations of Oxp or 5-FU with X-ray and X-ray alone resulted in high necrosis rates at later time points after treatment. Inhibition of apoptosis increased the amount of necrotic tumor cells, suggesting that a programmed form of necrosis can be induced by CT + X-ray. 5-FU and Oxp alone or in combination with X-ray and Irino plus X-ray were most effective in increasing the expression of RIP, IRF-5, and p53, proteins involved in necrotic and apoptotic cell death pathways. All treatments further resulted in the release of the immune activating danger signals high-mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70). The supernatants of the treated tumor cells induced maturation of dendritic cells. It is, therefore, concluded that combination of CT with X-ray is capable of inducing in vitro cell death forms of colorectal tumors with immunogenic potential.
... Recently, several proteins have been identified that sense extracellular nucleic acids and act as inducers of interferon (IFN) (Rock et al. 2011;Kawasaki et al. 2011). Normally, DNAse participates in degradation of inefficiently cleared CNAs released from dying cells (Gaipl et al. 2006). Deficiency of DNAse I, and the consequent inadequate removal of DNA from nuclear antigens, promotes susceptibility towards autoimmune disorders (Kawane et al. 2001, Kawane et al. 2006. ...
It has been estimated that 10(11) -10(12) cells, primarily of haematogenous origin, die in the adult human body daily, and a similar number is regenerated to maintain homeostasis. Despite the presence of an efficient scavenging system for dead cells, considerable amounts of fragmented genetic material enter the circulation in healthy individuals. Elevated blood levels of extracellular nucleic acids have been reported in various disease conditions; such as ageing and age-related degenerative disorders, cancer; acute and chronic inflammatory conditions, severe trauma and autoimmune disorders. In addition to genomic DNA and nucleosomes, mitochondrial DNA is also found in circulation, as are RNA and microRNA. There is extensive literature that suggests that extraneously added nucleic acids have biological actions. They can enter into cells in vitro and in vivo and induce genetic transformation and cellular and chromosomal damage; and experimentally added nucleic acids are capable of activating both innate and adaptive immune systems and inducing a sterile inflammatory response. The possibility as to whether circulating nucleic acids may, likewise, have biological activities has not been explored. In this review we raise the question as to whether circulating nucleic acids may have damaging effects on the host and be implicated in ageing and diverse acute and chronic human pathologies.
... They show particularly high affinity for nucleic acid (dsDNA) as well as for nuclear and cytoplasmic proteins that physically interact with nucleic acids, although individual reactivity patterns are highly heterogeneous. Autoantibody generation, in part originating from inappropriately processed self-antigens [1], is clearly T cell-dependent and occurs during germinal center reactions leading to a high degree of somatic hypermutation [2]. In these reactions, T-and B-cell targets can diversify by intra-and intermolecular epitope spreading, a mechanism in which crossreactivity and aberrant interactions between specific T-and B-cells favor the selection of self-reactive B-cells. ...
In human systemic lupus erythematosus (SLE), diverse autoantibodies accumulate over years before disease manifestation. Unaffected relatives of SLE patients frequently share a sustained production of autoantibodies with indiscriminable specificity, usually without ever acquiring the disease. We studied relations of IgG autoantibody profiles and peripheral blood activated regulatory T-cells (aTregs), represented by CD4(+)CD25(bright) T-cells that were regularly 70-90% Foxp3(+). We found consistent positive correlations of broad-range as well as specific SLE-associated IgG with aTreg frequencies within unaffected relatives, but not patients or unrelated controls. Our interpretation: unaffected relatives with shared genetic factors compensated pathogenic effects by aTregs engaged in parallel with the individual autoantibody production. To study this further, we applied a novel analytic approach named coreferentiality that tests the indirect relatedness of parameters in respect to multivariate phenotype data. Results show that independently of their direct correlation, aTreg frequencies and specific SLE-associated IgG were likely functionally related in unaffected relatives: they significantly parallelled each other in their relations to broad-range immunoblot autoantibody profiles. In unaffected relatives, we also found coreferential effects of genetic variation in the loci encoding IL-2 and CD25. A model of CD25 functional genetic effects constructed by coreferentiality maximization suggests that IL-2-CD25 interaction, likely stimulating aTregs in unaffected relatives, had an opposed effect in SLE patients, presumably triggering primarily T-effector cells in this group. Coreferentiality modeling as we do it here could also be useful in other contexts, particularly to explore combined functional genetic effects.
... Secreted "find-me" signals ensure the rapid recognition of apoptotic cells by macrophages. Failures in the clearance of the body's own dying cells have been demonstrated to activate the immune system and to contribute to the development of chronic autoimmunity [95]. The interaction of apoptotic cells with professional phagocytes, as macrophages are, is directly and indirectly mediated via the anionic phospholipid phosphatidylserine (PS) which becomes stably exposed on the outer cell membrane early in the apoptotic process (summarised in [96]). ...
Although cancer progression is primarily driven by the expansion of tumor cells, the tumor microenvironment and anti-tumor immunity also play important roles. Herein, we consider how tumors can become established by escaping immune surveillance and also how cancer cells can be rendered visible to the immune system by standard therapies such as radiotherapy or chemotherapy, either alone or in combination with additional immune stimulators. Although local radiotherapy results in DNA damage (targeted effects), it is also capable of inducing immunogenic forms of tumor cell death which are associated with a release of immune activating danger signals (non-targeted effects), such as necrosis. Necrotic tumor cells may result from continued exposure to death stimuli and/or an impaired phosphatidylserine (PS) dependent clearance of the dying tumor cells. In such circumstances, mature dendritic cells take up tumor antigen and mediate the induction of adaptive and innate anti-tumor immunity. Locally-triggered, systemic immune activation can also lead to a spontaneous regression of tumors or metastases that are outside the radiation field - an effect which is termed abscopal. Preclinical studies have demonstrated that combining radiotherapy with immune stimulation can induce anti-tumor immunity. Given that it takes time for immunity to develop following exposure to immunogenic tumor cells, we propose practical combination therapies that should be considered as a basis for future research and clinical practice. It is essential that radiation oncologists become more aware of the importance of the immune system to the success of cancer therapy.
... Clearance of apoptotic cells generally results in the activation of antiinflammatory signals, and thus may play an important role in the resolution of inflammation in mammals. Indeed it has been proposed that failure to clear apoptotic cells contributes to autoimmune diseases [8,9,10,11], and genes involved in apoptotic cell clearance have mutated splice variants in patients with systemic lupus erythematosus [12,13]. Genetic screens in Caenorhabditis elegans have identified many genes that participate in the clearance of apoptotic cells by neighbouring cells [14,15,16]. ...
Apoptosis, a genetically programmed cell death, allows for homeostasis and tissue remodelling during development of all multi-cellular organisms. Phagocytes swiftly recognize, engulf and digest apoptotic cells. Yet, to date the molecular mechanisms underlying this phagocytic process are still poorly understood. To delineate the molecular mechanisms of apoptotic cell clearance in Drosophila, we have carried out a deficiency screen and have identified three overlapping phagocytosis-defective mutants, which all delete the fly homologue of the ced-12 gene, known as Dmel\ced12. As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively. However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes. Using a nearby genetically interacting deficiency, we have found that the polycystic kidney disease 2 gene, pkd2, which encodes a member of the TRPP channel family, is also required for phagocytosis of apoptotic cells, thereby demonstrating a novel role for PKD2 in this process. We have also observed genetic interactions between pkd2, simu, drpr, rya-r44F, and retinophilin (rtp), also known as undertaker (uta), a gene encoding a MORN-repeat containing molecule, which we have recently found to be implicated in calcium homeostasis during phagocytosis. However, we have not found any genetic interaction between Dmel\ced-12 and simu. Based on these genetic interactions and recent reports demonstrating a role for the mammalian pkd-2 gene product in ER calcium release during store-operated calcium entry, we propose that PKD2 functions in the DRPR/RTP pathway to regulate calcium homeostasis during this process. Similarly to its C. elegans homologue, Dmel\Ced-12 appears to function in a genetically distinct pathway.
... This was very surprising, as polymorphisms in autophagy genes have been associated with an increased risk to develop inflammatory bowel disease. Furthermore, other studies have demonstrated that rapid clearance of apoptotic bodies is essential to prevent tissue inflammation [30] and that Atg5 general knockout mice showed a decreased removal of apoptotic cells and increased tissue inflammation [10]. However, the absence of tissue inflammation in unchallenged Atg7 IEC-KO mice might be reasoned by a minor role of autophagy in short living tissues such as the intestinal epithelial cell layer and the shedding of dead IECs into the gut lumen being unable to cause inflammation. ...
Genetic polymorphisms of autophagy-related genes have been associated with an increased risk to develop inflammatory bowel disease (IBD). Autophagy is an elementary process participating in several cellular events such as cellular clearance and nonapoptotic programmed cell death. Furthermore, autophagy may be involved in intestinal immune homeostasis due to its participation in the digestion of intracellular pathogens and in antigen presentation. In the present study, the role of autophagy in the intestinal epithelial layer was investigated. The intestinal epithelium is essential to maintain gut homeostasis, and defects within this barrier have been associated with the pathogenesis of IBD. Therefore, mice with intestinal epithelial deletion of Atg7 were generated and investigated in different mouse models. Knockout mice showed reduced size of granules and decreased levels of lysozyme in Paneth cells. However, this was dispensable for gut immune homeostasis and had no effect on susceptibility in mouse models of experimentally induced colitis.
... Inappropriate accumulation of secondary necrotic cells and cellular debris in the germinal centers of secondary lymph organs can lead to autoimmunity, and thus clearance defects are major players in the development of autoimmune diseases such as lupus. 85,86 It is also important to note that high-dose cyclosporine (50 mg/kg cyclosporine) to pretransplant donor-specific blood transfusion abrogated Tregs generation, whereas a lower dose (10 mg/kg) of cyclosporine promoted Tregs development either in synergy with perioperative donorspecific blood transfusion or by its own effect. 87 These data suggest that, at times, a lower dose of cyclosporine is more beneficial in patients with lupus and RA aimed at inducing Tregs. ...
Lupus is a chronic, systemic inflammatory condition in which eicosanoids, cytokines, nitric oxide (NO), a deranged immune system, and genetics play a significant role. Our studies revealed that an imbalance in the pro- and antioxidants and NO and an alteration in the metabolism of essential fatty acids exist in lupus. The current strategy of management includes administration of nonsteroidal anti-inflammatory drugs such as hydroxychloroquine and immunosuppressive drugs such as corticosteroids. Investigational drugs include the following: 1) belimumab, a fully human monoclonal antibody that specifically recognizes and inhibits the biological activity of B-lymphocyte stimulator, also known as B-cell-activation factor of the TNF family; 2) stem cell transplantation; 3) rituximab, a chimeric monoclonal antibody against CD20, which is primarily found on the surface of B-cells and can therefore destroy B-cells; and 4) IL-27, which has potent anti-inflammatory actions. Our studies showed that a regimen of corticosteroids and cyclophosphamide, and methods designed to enhance endothelial NO synthesis and augment antioxidant defenses, led to induction of long-lasting remission of the disease. These results suggest that methods designed to modulate molecular signatures of the disease process and suppress inflammation could be of significant benefit in lupus. Some of these strategies could be vagal nerve stimulation, glucose–insulin infusion, and administration of lipoxins, resolvins, protectins, and nitrolipids by themselves or their stable synthetic analogs that are known to suppress inflammation and help in the resolution and healing of the inflammation-induced damage. These strategies are likely to be useful not only in lupus but also in other conditions, such as rheumatoid arthritis, scleroderma, ischemia-reperfusion injury to the myocardium, ischemic heart disease, and sepsis.
... Deficiency in DNase I enzyme, and the resulting difficulty of removing DNA from nuclear antigens, promotes susceptibility to autoimmune disorders [7]. Failure of apoptosis leads to the development of a lupus-like syndrome [8]. DNase I deficiency was also found in patients with systemic lupus erythematosus (SLE), correlating with high titers of antibodies against nucleosomal antigens [9][10][11]. ...
Background and Aims. Deoxyribonuclease I (DNaseI) is an endonuclease that facilitates chromatin breakdown and promotes susceptibility to autoimmune disorders. The aim of current study was to investigate serum DNase I activity in patients with inflammatory bowel diseases (IBD). Patients and Methods. A cohort of 110 IBD patients was evaluated, aged 35 ± 12 years, 77 with Crohn's disease (CD) and 33 with ulcerative colitis (UC). 50 SLE patients and 50 healthy blood donors were examined as control groups. Results. DNase I activity in IBD patients was significantly lower than in healthy individuals, but higher than in SLE patients (P < .0001). Patients with UC showed higher DNase I activity than CD patients, P = .21. DNase I activity in female patients with IBD was significantly lower than in males, P = .024; however, no differences in DNase I activity were found in relation to gender in healthy individuals. DNase I activity has shown a strong negative correlation with the serum concentration of anti-nucleosomal antibodies in the autoimmune (SLE + IBD) cohort, as well as in the separate IBD cohort. Conclusions. Reduced serum DNase I activity probably has pathogenetic consequences in IBD. Induction of autoantibodies towards nucleosomes could be a reflection of impaired DNase I activity.
... IN VIVO-GENERATION AND GLOMERULAR DEPOSITION OF LARGE CHROMATIN FRAGMENTS-THE IMPACT OF RENAL DNASE-1 DOWNREGULATION AND UPREGULATION OF MMPS Reduced clearance of apoptotic cell debris has a causal role in necrotic transformation of apoptotic chromatin, and in chromatin deposition in tissue, such as glomeruli. [23][24][25][26][27][28] Yet, there is no apparent explanation as to why clearance of chromatin is reduced in SLE. ...
New information has profoundly improved our insight into the processes that account for lupus nephritis. This review summarizes the data proving that secondary necrotic chromatin fragments are generated and retained in kidneys at time-points when the major renal nuclease Dnase-1 is selectively and severely downregulated. Second, we discuss data, which may indicate that nuclease deficiencies are not associated with autoimmunity to chromatin. Secondary to downregulation of renal Dnase-1, large chromatin fragment-immunoglobulin G complexes are accumulated in glomerular basement membranes of patients producing anti-chromatin autoantibodies. Exposure of chromatin in situ in glomeruli is the factor that renders anti-chromatin (anti-dsDNA and anti-nucleosome) antibodies nephritogenic. Without exposed chromatin, they circulate as non-pathogenic antibodies. This shows that acquired loss of renal Dnase-1 enzyme activity is a dominant event responsible for the progression of lupus nephritis into end-stage disease. Before the loss of Dnase-1, lupus-prone (NZB × NZW) F1 mice develop mild or silent nephritis with mesangial immune complex deposits, which correlates solely with onset of anti-dsDNA antibody production. The principal cellular and molecular requirements needed to produce these autoantibodies have been explained experimentally, but the mechanism(s) accounting for them in vivo in context of lupus nephritis have not yet been determined. However, published data show that defects in nucleases operational in apoptotic or necrotic cell death are not associated with the induction of nephritogenic anti-dsDNA autoantibodies. The data discussed in this study explain how an unusual exposure of chromatin may be a central factor in the evolution of lupus nephritis in (NZB x NZW) F1 mice, but not in promoting nephritogenic chromatin-specific autoimmunity.
... Apoptotic cells are the unique source of nucleosomes, which are formed through cleavage of chromatin by nucleases [26][27][28]. At physiological condition, apoptotic cells, which maintain their membrane integrity for a certain time, have to be cleared quickly and eYciently by phagocytes in order to prevent the release of tissue-damaging intracellular constituents [29]. ...
The objective of the study is to determine whether the activity of DNase1 is associated to the presence of nephropathy in patients with SLE. Forty-five patients affected with SLE and renal involvement were analyzed. The type of renal involvement was type III or IV glomerulonephritis. At least two serum samples were withdrawn from each patient, one obtained in a renal flare and the other obtained in a period of clinical stability. C3 and C4 complement levels and anti-DNA antibodies were determined. DNase1 activity was measured using a radial enzyme-diffusion method. Results suggest that when comparison of DNase1 activity was established between samples obtained during a phase of active renal involvement and those obtained in the clinically stable phase, we did not find statistically significant differences. When the comparison was performed with matched samples of the same patient, DNase1 activity was lower when patients had active renal involvement than when samples were taken in clinically stable phase (21.21 μg/ml ± 16.47 vs. 25.62 μg/ml ± 18.81, p < 0.05). No difference in DNase1 activity was observed between samples positive or negative for anti-DNA antibodies. No difference in DNase1 activity was found in patients with normal or decreased levels of C3 (25.09 μg/ml ± 17.78 vs. 20.01 μg/ml ± 16.15, p = 0.073) or C4 (23.52 μm/ml ± 16.60 vs. 19.62 μg/ml ± 17.54, p = 0.060). We conclude that low DNase1 activity is associated to the active phase of type III or IV nephropathy. Therefore, it is possible that this enzyme plays an important role in the development of SLE nephropathy.
... Most importantly, the presence of ANA in healthy individuals (usually detectable in 5-10%) is very likely indicative of defective clearance of apoptotic cells in the body, which may also be genetically regulated [34]. Defective clearance of the apoptotic material leads to release of intracellular antigens, and, consequently, to the formation of autoantibodies, analogously to the mechanism responsible for the development of SLE [35]. ...
There is ample evidence demonstrating that accelerated atherosclerosis prevails in autoimmune rheumatic diseases, particularly in SLE, and that the risk is due not only to traditional cardiovascular risk factors but also to the disease itself. ANAs are a hallmark of SLE and are known even to antedate the development of SLE. Our aim was to investigate whether positive ANAs in young adults are associated with risk factors for atherosclerosis or subclinical markers of atherosclerosis.
ANAs were examined by IIF using HEp-2 cells as substrate in 2278 participants in the Cardiovascular Risk in Young Finns Study for whom detailed data on cardiovascular risk factors and markers of subclinical atherosclerosis (including brachial flow-mediated dilatation, carotid compliance and carotid intima-media thickness) were available.
In multivariate analyses, adjusted for age, BMI, serum concentrations of CRP, triglycerides, high-density lipoprotein and low-density lipoprotein cholesterol, blood pressure and smoking habits, ANA positivity (titre > 160) was inversely associated (beta = -0.145; P = 0.034) with carotid compliance in women.
Our results indicate that ANA positivity is associated with decreased carotid elasticity in women, suggesting that mechanisms resulting in ANA production may be involved in the development of early atherosclerosis.
Purpose: Previous investigations revealed influences of irradiation up to 2.0 Gy on the cytokine secretion profile of inflammatory, peritoneal mouse macrophages (pMФ). This raised the question if those alterations impact on dendritic cells and consecutive T-cell responses. Further, the impact of irradiation directly on pMФ capacity to induce T-cell responses was analyzed.
Materials and Methods: pMФ were LPS-activated, irradiated and the expression of activation markers was assessed. Treated pMФ were co-incubated with T-cells to investigate proliferation. To verify modulating properties of pMФ supernatants isolated 24 h after irradiation, bone marrow-derived dendritic cells (BMDC) were co-incubated with supernatants and activation markers as well as the BMDC-induced proliferation of T-cells were measured.
Results: pMФ showed a highly significantly decreased MHCII expression within a dose range from 0.7 to 2.0 Gy. Further, the proliferation rate of CD4⁺ T-cells was decreased after co-incubation with in particular 2.0 Gy irradiated pMФ. The co-incubation of BMDC with supernatants of activated, irradiated pMФ significantly reduced the CD40 expression, but did not impact on the BMDC-derived induction of T-cell proliferation.
Conclusions: Inflammatory macrophages being exposed to irradiation have the potential to modulate consecutive adaptive immune reactions. But supernatants of irradiated macrophages do not influence the DC-mediated induction of T cell proliferation.
Radiotherapy (RT) predominantly is aimed to induce DNA damage in tumour cells that results in reduction of their clonogenicity and finally in tumour cell death. Adaptation of RT with higher single doses has become necessary and led to a more detailed view on what kind of tumour cell death is induced and which immunological consequences result from it. RT is capable of rendering tumour cells immunogenic by modifying the tumour cell phenotype and the microenvironment. Danger signals are released as well as the senescence-associated secretory phenotype. This results in maturation of dendritic cells and priming of cytotoxic T cells as well as in activation of natural killer cells. However, RT on the other hand can also result in immune suppressive events including apoptosis induction and foster tumour cell proliferation. That’s why RT is nowadays increasingly combined with selected immunotherapies.
Inefficient clearance of apoptotic cells and the subsequent exposure of the immune system to nuclear contents are crucially involved in the pathogenesis of systemic lupus erythematosus (SLE). Factor VII-activating protease (FSAP) is activated in serum upon contact with dead cells, and releases nucleosomes from late apoptotic cells into the extracellular environment. We investigated whether FSAP-mediated nucleosome release from late apoptotic cells is affected in SLE patients. Nucleosome release in sera of 27 SLE patients and 30 healthy controls was investigated by incubating late apoptotic Jurkat cells with serum and analyzing the remaining DNA content by flow cytometry. We found that nucleosome release in sera of SLE patients with high disease activity was significantly decreased when compared with that in SLE sera obtained during low disease activity or from healthy individuals. Upon removal of IgG/IgM antibodies from SLE sera, nucleosome release was restored. Similarly, monoclonal anti-nuclear antibodies inhibited nucleosome release in healthy donor serum or by plasma-purified FSAP. This inhibition was lost when Fab fragments were used, suggesting that antigen crosslinking is involved. In conclusion, FSAP-mediated nucleosome release from late apoptotic cells is greatly impaired in SLE patient sera, possibly hampering the clearance of these cells and thereby propagating inflammation. This article is protected by copyright. All rights reserved.
Classical antitumoral therapies are basically perceived as a direct and lytic action of the chosen therapeutic agent against tumor cells. Nevertheless, growing evidence indicates that some anticancer strategies can work through the action of the immune system. Imatinib Mesylate (IM), a targeted drug, helped us to unravel some mechanisms of action of a natural killer sub-population: the "Interferon-producing Killer dendritic Cells" (IKDC). Thoses cells possess anti-tumoral lytic capacities, synergistically amplified by IM and interleukine (IL)-2, under the dependency of IL-15, and also antigen-presenting capability. Those cells are capable of TRAIL-mediated lysis and of antigen uptake, processing and presentation to naives T cells.
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with various clinical manifestations affecting different tissues. A characteristic feature of SLE is the presence of autoantibodies against double-stranded (ds)DNA, histones and nucleosomes, and other chromatin components. SLE is a prototype type III hypersensitivity reaction. Local deposition of anti-nuclear antibodies in complex with released chromatin induces serious inflammatory conditions by activation of the complement system. The severe renal manifestation, lupus nephritis, is classified based on histological findings in renal biopsies. Apoptotic debris, including chromatin, is present in the extracellular matrix and circulation of patients with SLE. This may be due to an aberrant process of apoptosis and/or insufficient clearance of apoptotic cells/chromatin. The non-cleared apoptotic debris may lead to activation of both the innate and adaptive immune systems. In addition, an aberrant presentation of peptides by antigen-presenting cells, disturbed selection processes for lymphocytes, and deregulated lymphocyte responses may be involved in the development of autoimmunity. In the present review, we briefly will summarize current knowledge on the pathogenesis of SLE. We will also critically discuss and challenge central issues that need to be addressed in order to fully understand the pathogenic mechanisms involved in the development of SLE and in order to have an improved diagnosis for SLE. Disappointingly, in our opinion, there are still more questions than answers for the pathogenesis, diagnosis, and treatment of SLE.
Apoptotic nucleosomes are structurally and immunologically involved in lupus nephritis. The purpose of this study was to examine the expression and function of laminins and their interactions with nucleosomes in the kidneys of patients with lupus nephritis, using surface plasmon resonance (SPR) analysis.
SPR interaction analysis was used to quantify the strength of laminin-nucleosome interactions. Electron microscopy techniques were used to determine in vivo colocalization of IgG, chromatin, and laminin β1, as well as to characterize nucleosome-laminin interactions in vitro.
Nucleosomes were found to possess high affinity for laminin β1-containing laminins and to have the potential to form stable molecular complexes with these structures. In vivo, laminin β1 was aberrantly expressed in the glomerular basement membrane (GMB) of lupus nephritis patients, and in situ, it acted as a nucleosome ligand, selectively colocalizing with nucleosomes within electron-dense deposits (EDDs). Normal adult laminin 11, which contains laminin β2, did not bind nucleosomes, and it did not colocalize in vivo with the nucleosomes in the nephritic GBM. In addition, TGFβ1 was expressed by the glomerular mesangium, glomerular endothelial cells, and by podocytes in patients with lupus nephritis. It was trapped in situ within EDDs by an as-yet-unknown ligand. As was recently described in a transgenic mouse model, paracrine kidney glomerular TGFβ1 may thereby contribute to the development of glomerulopathy via the induction of laminin β1 incorporation in the GBM, whereas systemic blood vessel-derived TGFβ1 could be trapped during filtration.
Our findings of the specific high-affinity binding of nucleosomes to aberrantly expressed laminin β1 in the GBM and their colocalization in the GBM may explain important features of the initial steps in the pathogenesis of lupus nephritis, the planted antigen hypothesis.
Recent findings show that transformation of mild glomerulonephritis into end-stage disease coincides with shutdown of renal DNaseI expression in (NZBxNZW)F1 mice. Down-regulation of DNaseI results in reduced chromatin fragmentation and deposition of extracellular chromatin fragments in glomerular basement membranes where they appear in complex with IgG antibodies. Here, we implicate the anti-apoptotic and survival protein, tumor necrosis factor receptor-associated protein 1 (Trap1) in the disease process, based on the observation that annotated transcripts from this gene overlap with transcripts from the DNaseI gene. Furthermore, we translate these observations to human lupus nephritis. In this study, mouse and human DNaseI and Trap1 mRNA levels were determined by quantitative PCR and compared with protein expression levels and clinical data. Cellular localization was analyzed by immune electron microscopy, IHC, and in situ hybridization. Data indicate that silencing of DNaseI gene expression correlates inversely with expression of the Trap1 gene. Our observations suggest that the mouse model is relevant for the aspects of disease progression in human lupus nephritis. Acquired silencing of the renal DNaseI gene has been shown to be important for progression of disease in both the murine and human forms of lupus nephritis. Early mesangial nephritis initiates a cascade of inflammatory signals that lead to up-regulation of Trap1 and a consequent down-regulation of renal DNaseI by transcriptional interference.
Background:
Decreased activity of serum desoxyribonuclease I (DNase I) in systemic lupus erythematosus (SLE) has been reported, but its role as a biomarker in SLE is still unelucidated.
Methods:
Seventy-seven SLE patients (aged 39.6 ± 13.1 years) were studied for serum DNase I activity, levels of antinuclear (ANA), anti-dsDNA [high-avidity ELISA, conventional ELISA and indirect immunofluorescence (IIF)], anti-nucleosome, anti-histone antibodies, complement components C3 and C4. SLE disease activity was evaluated by disease activity index (SLEDAI-2K). Thirty-five patients were serologically and clinically followed for 3-12 months (mean 5.6 ± 2.8). Thirty-seven healthy blood donors were the control group.
Results:
DNase I activity in SLE patients was lower than in healthy controls (p<0.01). DNase I activity was in positive correlation with SLEDAI-2K (p<0.01), levels of ANA, anti-dsDNA, anti-nucleosome and anti-histone antibodies (p<0.01) and in negative correlation with C3 concentration (p<0.05). The highest correlation was found between DNase I activity and anti-dsDNA concentrations determined by high-avidity ELISA (r=0.624), followed by IIF (r=0.541) and conventional ELISA (r=0.405). In the follow-up study, DNase I activity also correlated with SLEDAI-2K (p<0.01). SLE patients with low DNase I activity more frequently had SLE-specific cutaneous lesions (p<0.05).
Conclusions:
Monitoring of DNase I activity simultaneously with SLEDAI-2K might be a useful tool in the follow-up of SLE. An increase of DNase I activity characterized relapse in most SLE patients, although it did not reach the levels of healthy individuals. A decrease of DNase I activity in SLE flare-ups might be a functional biomarker of a subset of patients with specific dysfunction of apoptotic chromatin degradation.
Antineutrophil cytoplasmic antibodies (ANCAs) target proteins normally retained within neutrophils, indicating that cell death is involved in the autoimmunity process. Still, ANCA pathogenesis remains obscure. ANCAs activate neutrophils inducing their respiratory burst and a peculiar form of cell death, named NETosis, characterized by formation of neutrophil extracellular traps (NETs), decondensed chromatin threads decorated with cytoplasmic proteins endorsed with antimicrobial activity. NETs have been consistently detected in ANCA-associated small-vessel vasculitis, and this association prompted us to test whether the peculiar structure of NET favors neutrophil proteins uploading into myeloid dendritic cells and the induction of ANCAs and associated autoimmunity. Here we show that myeloid DCs uploaded with and activated by NET components induce ANCA and autoimmunity when injected into naive mice. DC uploading and autoimmunity induction are prevented by NET treatment with DNAse, indicating that NET structural integrity is needed to maintain the antigenicity of cytoplasmic proteins. We found NET intermingling with myeloid dendritic cells also positive for neutrophil myeloperoxidase in myeloperoxidase-ANCA-associated microscopic poliangiitis providing a potential correlative picture in human pathology. These data provide the first demonstration that NET structures are highly immunogenic such to trigger adaptive immune response relevant for autoimmunity.
We have demonstrated that glomerular expression of polyomavirus large T antigen (T-ag) in a binary tetracycline-regulated T-ag transgenic mouse model (i) terminated tolerance for nucleosomes, (ii) released complexes of nucleosomes and T-ag to the microenvironment from dead cells, and (iii) that these complexes bound induced anti-nucleosome antibodies and finally (iv) that they associated with glomerular membranes as immune complexes. This process may be relevant for human lupus nephritis, since productive polyomavirus infection is associated with this organ manifestation. Here, we compare nephritis in the T-ag transgenic mouse with nephritis in human SLE. Glomerular sections were analysed by transmission electron microscopy, immune electron microscopy (IEM) and by co-localization IEM and TUNEL IEM assays to compare morphological changes, composition of immune complexes and formation of nucleosome–T-ag complexes. Affinity of nucleosome–T-ag complexes for glomerular collagen IV and laminin was determined by surface plasmon resonance (SPR). Analyses revealed electron dense structures in both human and murine kidney samples. These EDS were shown to contain T-ag, DNA and histones, indicating that extra-cellular chromatin may originate from polyomavirus infected cells in human kidneys. SPR analyses demonstrated high affinity of nucleosomes and nucleosome–T-ag complexes for collagen IV and laminin. Complexes of nucleosomes, T-ag and anti-T-ag and anti-dsDNA antibodies bind glomerular membranes and contribute to the evolution of lupus nephritis in human SLE.
Squamous cell carcinoma (SCC) accounts for more than 90% of malignant tumors of the oral cavity. In Taiwan, oral squamous cell carcinoma (OSCC) is among the most frequent malignancies, largely due to betal quid chewing. Despite the recent improvement in treatment results, the long-term outcome of OSCC generally remains poor, especially for those with advanced diseases. It is therefore desirable to identify potential biomarkers that may aid in risk stratification and perhaps the development of therapeutic targets. In this study, we exploited two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry to compare the proteome maps of 10 OSCC specimens with their adjacent nontumorous epithelia to identify differentially expressed proteins. Comparative proteomics indicated that 17 proteins were differentially expressed in OSCC with 11 up-regulated and 6 down-regulated proteins. These deregulated proteins participated in cytoskeletal functions, cell signaling, antiapoptosis, angiogenesis, lipid metabolism, drug metabolism, and protein translation/turnover. They were all associated with tumor development in various cancers. Among the dys-regulated proteins, the immunoexpression of three proteins including nicotinamide N-methyltransferase, apolipoprotein AI, and 14-3-3 zeta were evaluated in 38 OSCCs of testing cohort to confirm the proteomics data. Subsequently, the expression of 14-3-3 zeta, as the most relevant to OSCC progression determined by testing cohort, was further assessed in 80 OSCCs of independent validation cohort to identify the clinical relevance of its expression. By this comprehensive study, we identified 14-3-3 zeta as the only prognosticator of local recurrence-free survival (LRFS) and also an independently predicted factor of disease-specific survival (DSS).
To assess the relationship between serum C3 or C4 levels and lupus renal flare, C3 and C4 levels were measured bimonthly in 71 lupus nephritis patients for a mean of 35 months, during which time 70 renal flares were identified. Comparing baseline, pre-flare, and at-flare values indicated that neither C3 nor C4 levels decreased pre-flare, but both decreased on average significantly at flare. However, sensitivity/specificity for C3 (75%/71%) and C4 (48%/71%) were low. To account for other influencing factors, multiple regression was performed that included bimonthly values of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), and genotype data on C3 (S/F), CRP (1846G > A), and the complement regulator factor H (Y402H). This analysis revealed that reduced levels of C4, but not C3, were independently associated with the two-month pre-flare period. Conversely, reduced levels of C3, but not C4, were independently associated with the flare visit. Significant pro-flare interactions included low C3 levels with the factor H 402HH-encoding genotype, and low CRP levels with the C3 F allele. Together these data suggest that C4 activation is critical for initiating renal flare while C3 activation is involved in the actual tissue damage, and that these effects are influenced by genetic variability in complement activation and regulation.
Asthma and chronic obstructive pulmonary disease (COPD) represent increasingly common respiratory conditions with a clear unmet need for more effective and safer therapy. Airway inflammation is key to both asthma pathogenesis and exacerbation of symptoms in COPD. Several lines of evidence are now emerging, demonstrating that, in addition to their established effectiveness in the treatment of atherosclerotic disease, statins also exhibit anti-inflammatory properties, which may be of relevance for the treatment of chronic lung disease, including asthma and COPD. This review will examine the diverse in vitro and in vivo anti-inflammatory properties of statins and consider the available evidence that statins represent novel therapeutic interventions for asthma and COPD.
Calreticulin (CRT), when localized to the endoplasmic reticulum (ER), has important functions in directing proper conformation of proteins and glycoproteins, as well as in homeostatic control of cytosolic and ER calcium levels. There is also steadily accumulating evidence for diverse roles for CRT localized outside the ER, including data suggesting important roles for CRT localized to the outer cell surface of a variety of cell types, in the cytosol, and in the extracellular matrix (ECM). Furthermore, the addition of exogenous CRT rescues numerous CRT-driven functions, such as adhesion, migration, phagocytosis, and immunoregulatory functions of CRT-null cells. Recent studies show that topically applied CRT has diverse and profound biological effects that enhance cutaneous wound healing in animal models. This evidence for extracellular bioactivities of CRT has provided new insights into this classically ER-resident protein, despite a lack of knowledge of how CRT exits from the ER to the cell surface or how it is released into the extracellular milieu. Nonetheless, it has become clear that CRT is a multicompartmental protein that regulates a wide array of cellular responses important in physiological and pathological processes, such as wound healing, the immune response, fibrosis, and cancer.-Gold, L. I., Eggleton, P., Sweetwyne, M. T., Van Duyn, L. B., Greives, M. R., Naylor, S.-M., Michalak, M., Murphy-Ullrich, J. E. Calreticulin: non-endoplamic reticulum functions in physiology and disease.
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 TNFalpha and IL-1beta after contact with ITC plus AnxA5 in comparison with ITC alone, while the amount of TGF-beta 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.
Immunoprecipitable double-stranded (dsDNA) was previously shown to persist in the circulation of a clinically recognizable subgroup of patients with systemic lupus erythematosus (SLE). Plasma from 10 such patients was subjected to a DNA isolation procedure that used a combination of proteolysis, phenol extraction, and hydroxylapatite adsorption and elution in the presence of urea. The isolated dsDNA was radiolabeled by nick translation and then characterized by isopyknic ultracentrifugation in CsCl under both neutral and alkaline conditions, as well as after digestion with S1-endonuclease. These experiments demonstrated essential identity in nucleotide base composition between the plasma-derived DNA and human genomic DNA. The presence of specific human base sequences in the plasma DNA was demonstrated by finding that authentic human genomic DNA accelerated the renaturation of plasma DNA when compared with the effect of nonhuman, control DNA. The proportion of such sequences in plasma DNA was estimated by attempting to renature the plasma DNA in the presence of human DNA under conditions shown to result in complete renaturation of human DNA in model experiments. In this way, a minimum of 47% of plasma DNA base sequences could be shown also to be present in human genomic DNA. However, an average of 10-20% of the plasma-derived DNA failed to renature under these conditions, a result that was further confirmed by comparing the renaturation of the tritium-labeled plasma DNA specimens, in double-label experiments, with internal controls consisting of 14C-labeled authentic human DNA. Attempts to drive the reaction to completion with human DNA led to a similar conclusion. The relative nonrenaturability of this fraction of plasma DNA did not appear to be attributable to extensive chain breakage, although adequate analysis of this DNA subfraction was limited by reagent availability. It was therefore concluded that, in this group of SLE patients, persistently circulating DNA consisted largely of base sequences also found in human genomic DNA. The additional presence in plasma of a DNA subfraction that differed in its renaturation behavior from human genomic DNA was recognized, although its significance could not be established with certainty.
Using the in vitro DNA labeling technique of nick translation on purified plasma DNA, we have estimated the plasma DNA concentration in three normal individuals to be 266 +/- 57 ng/ml (mean +/- SD). This was not significantly different in three patients with a chronic inflammatory disease (209 +/- 14 ng/ml) or in five patients with steroid-inactivated systemic lupus erythematosus (SLE) (293 +/- 57 ng/ml). In two untreated, newly diagnosed, active SLE patients, however, the plasma DNA concentration was considerably higher (4,024 and 2,437 ng/ml, respectively). Characterization of these in vitro labeled DNA preparations by neutral sucrose-gradient sedimentation analysis showed a sedimentation coefficient of 6-8S, corresponding to a molecular weight of similar to or approximately 0.2-0.45 x 10(6). No difference was observed between normal subjects or patients. In addition, the relative size uniformity of these DNA molecules might suggest some form of specific protection of the DNA from blood DNAases. Further characterization in terms of buoyant density in cesium chloride did not reveal a difference between normal or SLE plasma and the human (HEp-2 cell) DNA used as marker. Taking into account the limitations of the method, no indication of a possible exogenous origin of the DNA circulating in SLE patients could be found. The physiological or pathophysiological role of this plasma DNA remains to be determined.
Immune context is an essential determinant of the host response to potential autoantigens. The clustering of the autoantigens targeted in systemic lupus erythematosus within surface blebs of apoptotic cells generates high concentrations of autoantigen within discrete subcellular packages. We demonstrate here that when apoptosis is induced by Sindbis virus infection, viral antigens and autoantigens cocluster exclusively in small surface blebs of apoptotic cells. The surface of these blebs is rich in viral glycoproteins, and virions can be seen blebbing from their surface. We propose that these blebs of mixed foreign and self-origin define a novel immune context that may challenge self-tolerance.
Apoptotic cell death is important in the development and homeostasis of multicellular organisms and is a highly controlled means of eliminating dangerous, damaged or unnecessary cells without causing an inflammatory response or tissue damage,. We now show that the presence of apoptotic cells during monocyte activation increases their secretion of the anti-inflammatory and immunoregulatory cytokine interleukin 10 (IL-10) and decreases secretion of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), IL-1 and IL-12. This may inhibit inflammation and contribute to impaired cell-mediated immunity in conditions associated with increased apoptosis, such as viral infections, pregnancy, cancer and exposure to radiation.
The complement system plays a paradoxical role in the development and expression of autoimmunity in humans. The activation of complement in systemic lupus erythematosus (SLE) contributes to tissue injury. In contrast, inherited deficiency of classical pathway components, particularly C1q (ref. 1), is powerfully associated with the development of SLE. This leads to the hypothesis that a physiological action of the early part of the classical pathway protects against the development of SLE (ref. 2) and implies that C1q may play a key role in this respect. C1q-deficient (C1qa-/-) mice were generated by gene targeting and monitored for eight months. C1qa-/- mice had increased mortality and higher titres of autoantibodies, compared with strain-matched controls. Of the C1qa-/- mice, 25% had glomerulonephritis with immune deposits and multiple apoptotic cell bodies. Among mice without glomerulonephritis, there were significantly greater numbers of glomerular apoptotic bodies in C1q-deficient mice compared with controls. The phenotype associated with C1q deficiency was modified by background genes. These findings are compatible with the hypothesis that C1q deficiency causes autoimmunity by impairment of the clearance of apoptotic cells.
During apoptotic cell death, cell surface ligands initiate phagocytosis of the dying cell. Clearance of these apoptotic cells is thought to occur without an immune response. Since a number of autoantigens are located at the cell surface or within apoptotic blebs, we examined whether exposure of mice to syngeneic apoptotic cells by the intravenous route could induce autoantibody production. Normal mice injected with syngeneic apoptotic thymocytes developed antinuclear autoantibodies and anticardiolipin and anti-ssDNA antibodies. The autoantibody levels were generally lower than those observed in MRL/Faslpr mice and were transient. Surprisingly, six out of six immunized mice demonstrated immunoglobulin G deposition in the glomeruli several months after immunization. These findings indicate that systemic exposure to apoptotic cells can induce an immune response in normal mice, and may help to explain antigen selection and initiation of the immune response in diseases characterized by increased rates of apoptosis such as AIDS and, possibly, systemic lupus erythematosus.
Apoptotic cells are rapidly engulfed by phagocytes, but the receptors and ligands responsible for this phenomenon are incompletely characterized. Previously described receptors on blood- derived macrophages have been characterized in the absence of serum and show a relatively low uptake of apoptotic cells. Addition of serum to the phagocytosis assays increased the uptake of apoptotic cells by more than threefold. The serum factors responsible for enhanced uptake were identified as complement components that required activation of both the classical pathway and alternative pathway amplification loop. Exposure of phosphatidylserine on the apoptotic cell surface was partially responsible for complement activation and resulted in coating the apoptotic cell surface with C3bi. In the presence of serum, the macrophage receptors for C3bi, CR3 (CD11b/CD18) and CR4 (CD11c/CD18), were significantly more efficient in the uptake of apoptotic cells compared with previously described receptors implicated in clearance. Complement activation is likely to be required for efficient uptake of apoptotic cells within the systemic circulation, and early component deficiencies could predispose to systemic autoimmunity by enhanced exposure to and/or aberrant deposition of apoptotic cells.
Although different macrophages exploit different cell surface receptors to recognize apoptotic lymphocytes, indirect evidence suggested that the phosphatidylserine (PS) that appears on the surface of lymphocytes undergoing apoptosis participates in specific recognition by all types of macrophages. To test this possibility directly, annexin V, a protein that specifically binds to PS, was used to mask this phospholipid on the apoptotic cell surface. Preincubation of apoptotic lymphocytes with annexin V blocked phagocytosis by elicited mouse peritoneal macrophages, macrophages of the mouse J774 cell line and mouse bone marrow macrophages. Similarly, annexin V was able to inhibit phagocytosis of lipid-symmetric erythrocytes, another target cell upon which PS is exposed. Together these results demonstrate directly that macrophages of all types depend on the PS exposed on the surface of apoptotic lymphocytes for recognition and phagocytosis.
Exposure of phosphatidylserine on the outer leaflet of the plasma membrane is a surface change common to many apoptotic cells. Normally restricted to the inner leaflet, phosphatidylserine appears as a result of decreased aminophospholipid translocase activity and activation of a calcium-dependent scramblase. Phosphatidylserine exposure has several potential biological consequences, one of which is recognition and removal of the apoptotic cell by phagocytes. It is still not clear which receptors mediate PS recognition on apoptotic cells; however, several interesting candidates have been proposed. These include the Class B scavenger and thrombospondin receptor CD36, an oxLDL receptor (CD68), CD14, annexins, beta2 glycoprotein I, gas-6 and a novel activity expressed on macrophages stimulated with digestible particles such as beta-glucan. Whether PS is the sole ligand recognized by phagocytes or whether it associated with other molecules to form a complex ligand remains to be determined.
Serum amyloid P component (SAP), a highly conserved plasma protein named for its universal presence in amyloid deposits, is the single normal circulating protein that shows specific calcium-dependent binding to DNA and chromatin in physiological conditions. The avid binding of SAP displaces H1-type histones and thereby solubilizes native long chromatin, which is otherwise profoundly insoluble at the physiological ionic strength of extracellular fluids. Furthermore, SAP binds in vivo both to apoptotic cells, the surface blebs of which bear chromatin fragments, and to nuclear debris released by necrosis. SAP may therefore participate in handling of chromatin exposed by cell death. Here we show that mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoimmunity and severe glomerulonephritis, a phenotype resembling human systemic lupus erythematosus, a serious autoimmune disease. The SAP-/- mice also have enhanced anti-DNA responses to immunization with extrinsic chromatin, and we demonstrate that degradation of long chromatin is retarded in the presence of SAP both in vitro and in vivo. These findings indicate that SAP has an important physiological role, inhibiting the formation of pathogenic autoantibodies against chromatin and DNA, probably by binding to chromatin and regulating its degradation.
Systemic autoimmune diseases are a genetically complex, heterogeneous group of disorders in which the immune system targets a diverse but highly specific group of intracellular autoantigens. The molecules targeted are not unified by common structure, function, or distribution in control cells but become clustered and concentrated in surface blebs when cells undergo apoptosis. We show here that the majority of autoantigens targeted across the spectrum of human systemic autoimmune diseases are efficiently cleaved by granzyme B in vitro and during cytotoxic lymphocyte granule-induced death, generating unique fragments not observed during any other form of apoptosis. These molecules are not cleaved by caspase-8, although this protease has a very similar specificity to granzyme B. The granzyme B cleavage sites in autoantigens contain amino acids in the P(2) and P(3) positions that are preferred by granzyme B but are not tolerated by caspase-8. In contrast to autoantigens, nonautoantigens are either not cleaved by granzyme B or are cleaved to generate fragments identical to those formed in other forms of apoptosis. The striking ability of granzyme B to generate unique fragments is therefore an exclusive property of autoantigens and unifies the majority of molecules targeted in this spectrum of diseases. These results focus attention on the role of the cytotoxic lymphocyte granule-induced death pathway in the initiation and propagation of systemic autoimmunity.
Recombinant annexin V (rAnV) has been used to identify apoptotic cells based on its ability to bind phosphatidylserine (PS), a lipid normally restricted to the cytoplasmic face of the plasma membrane, but externalized early during apoptosis. However, this association of rAnV binding and apoptosis is not an obligatory one. We demonstrate that rAnV binds to a large fraction of murine B cells bearing selectable Ag receptors despite the fact that these cells are not apoptotic. Phosphatidylserine, which is uniformly distributed on resting B cells, is mobilized to co-cap with IgM on anti-IgM-treated B cells and to colocalize with GM1, a marker of lipid rafts. Cross-linking PS before anti-IgM treatment sequesters this lipid and alters signaling through IgM. Thus, PS exposed on the majority of B cells in vivo does not reflect early apoptosis, but, instead, plays a role in receptor-mediated signaling events.
Serum immunoglobulin (Ig)M provides the initial response to foreign antigen and plays a regulatory role in subsequent immune response development, accelerating the production of high-affinity IgG. Here we show that mice deficient in serum IgM have an increased propensity to spontaneous autoimmunity as judged by the development with age of serum IgG anti-DNA antibodies and the renal deposition of IgG and complement. They also exhibit augmented anti-DNA IgG production on exposure to lipopolysaccharide. Thus, deficiency in serum IgM leads to diminished responsiveness to foreign antigens but increased responsiveness to self—a paradoxical association reminiscent of that described in humans deficient in complement or IgA.
We wondered whether serum IgM might play an analogous role with regard to the response to self-antigens. However, here—in contrast to the sluggish response to foreign antigens—we find that deficiency in serum IgM actually predisposes to the development of IgG antibodies to autoantigens.
Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that affects over one million people in the United States. SLE is characterized by the presence of anti-nuclear antibodies (ANA) directed against naked DNA and entire nucleosomes. It is thought that the resulting immune complexes accumulate in vessel walls, glomeruli and joints and cause a hypersensitivity reaction type III, which manifests as glomerulonephritis, arthritis and general vasculitis. The aetiology of SLE is unknown, but several studies suggest that increased liberation or disturbed clearance of nuclear DNA-protein complexes after cell death may initiate and propagate the disease. Consequently, Dnase1, which is the major nuclease present in serum, urine and secreta, may be responsible for the removal of DNA from nuclear antigens at sites of high cell turnover and thus for the prevention of SLE (refs 7-11). To test this hypothesis, we have generated Dnase1-deficient mice by gene targeting. We report here that these animals show the classical symptoms of SLE, namely the presence of ANA, the deposition of immune complexes in glomeruli and full-blown glomerulonephritis in a Dnase1-dose-dependent manner. Moreover, in agreement with earlier reports, we found Dnase1 activities in serum to be lower in SLE patients than in normal subjects. Our findings suggest that lack or reduction of Dnase1 is a critical factor in the initiation of human SLE.
Cells generally maintain an asymmetric distribution of phospholipids across the plasma membrane bilayer, restricting the phospholipid, phosphatidylserine (PS), to the inner leaflet of the plasma membrane. When cells undergo apoptosis, this asymmetric transbilayer distribution is lost, bringing PS to the surface where it acts as a signal for engulfment by phagocytes. The fluorescent dye merocyanine 540 specifically stains the plasma membrane of apoptotic cells which have lost their asymmetric distribution of phospholipids. However, it also stains non-apoptotic macrophages, suggesting that phospholipid asymmetry may not be maintained in these cells, and thus that they may express PS on their surface. Here, the PS-binding protein, annexin V, was used to show that in fact normal macrophages do express PS on their surface. Furthermore, pre-treating macrophages with annexin V was found to inhibit phagocytosis of apoptotic thymocytes and thymocytes on which PS expression was artificially induced, but did not inhibit phagocytosis of latex beads or Fc receptor-mediated phagocytosis of opsonized erythrocytes. These results indicate that PS is constitutively expressed on the surface of macrophages and is functionally significant for the phagocytosis of PS-expressing target cells.
The strongest susceptibility genes for the development of systemic lupus erythematosus (SLE) in humans are null mutants of classical pathway complement proteins. There is a hierarchy of disease susceptibility and severity according to the position of the missing protein in the activation pathway, with the severest disease associated with C1q deficiency. Here we demonstrate, using novel in vivo models of apoptotic cell clearance during sterile peritonitis, a similar hierarchical role for classical pathway complement proteins in vivo in the clearance of apoptotic cells by macrophages. Our results constitute the first demonstration of an impairment in the phagocytosis of apoptotic cells by macrophages in vivo in a mammalian system. Apoptotic cells are thought to be a major source of the autoantigens of SLE, and impairment of their removal by complement may explain the link between hereditary complement deficiency and the development of SLE.
Pentraxins are acute-phase proteins produced in vivo during inflammatory reactions. Classical short pentraxins, C-reactive protein, and serum amyloid P component are generated in the liver in response to interleukin (IL)-6. The long pentraxin PTX3 is produced in tissues under the control of primary proinflammatory signals, such as lipopolysaccharide, IL-1 beta, and tumor necrosis factor-alpha, which also promote maturation of dendritic cells (DCs). Cell death commonly occurs during inflammatory reactions. In this study, it is shown that PTX3 specifically binds to dying cells. The binding was dose dependent and saturable. Recognition was restricted to extranuclear membrane domains and to a chronological window after UV irradiation or after CD95 cross-linking-induced or spontaneous cell death in vitro. PTX3 bound to necrotic cells to a lesser extent. Human DCs failed to internalize dying cells in the presence of PTX3, while they took up normally soluble or inert particulate substrates. These results suggest that PTX3 sequesters cell remnants from antigen-presenting cells, possibly contributing to preventing the onset of autoimmune reactions in inflamed tissues. (Blood. 2000;96:4300-4306)
Recombinant annexin V (rAnV) has been used in flow cytometry to identify cells undergoing apoptosis, based on its ability to bind to phosphatidylserine, a negatively charged lipid normally restricted to the cytoplasmic face of the plasma membrane but externalized early during apoptosis. When we stained murine bone marrow (BM) cells with fluorescently labeled rAnV, we found that a surprisingly large fraction of BM B cells bearing selectable transgenic Ag receptors bind significant amounts of rAnV, but that these cells are not apoptotic. Here, we show that binding of rAnV to developing B cells in normal mice correlates with B cell receptor-dependent selection events at several stages of development within both B-1 and B-2 cell subsets. In fact, nearly all B-1 B cells and splenic marginal zone B cells bind rAnV, suggesting that the externalization of phosphatidylserine occurs once mature B cells are selected through BCR-mediated signaling. However, this plasma membrane alteration is apparently not shared by all lymphocytes, because we did not find a parallel population of rAnV-binding viable T cells in vivo in normal or TCR transgenic mice. We also show that BM stromal cell lines can influence the extent of rAnV binding by viable BM B cells during coculture in vitro. We suggest that rAnV detects a potentially important membrane alteration that occurs as B cells develop in the BM and are readied for export to the peripheral lymphoid organs and again among mature B cells recruited to the marginal zone or the B-1 compartment.
Surfactant protein A (SP-A) is an innate immune molecule that binds foreign organisms that invade the lungs and targets them for phagocytic clearance by the resident pulmonary phagocyte, the alveolar macrophage (AM). We hypothesized that SP-A binds to and enhances macrophage uptake of other nonself particles, specifically apoptotic polymorphonuclear neutrophils (PMNs). PMNs are recruited into the lungs during inflammation, but as inflammation is resolved, PMNs undergo apoptosis and are phagocytosed by AMs. We determined that SP-A increases AM phagocytosis of apoptotic PMNs 280 +/- 62% above the no protein control value. The increase is dose dependent, and heat-treated SP-A still enhanced uptake, whereas deglycosylated SP-A had significantly diminished ability to enhance phagocytosis. Surfactant protein D also increased phagocytosis of apoptotic PMNs by approximately 125%. However, other proteins that are structurally homologous to SP-A, mannose-binding lectin and complement protein 1q, did not. SP-A enhances phagocytosis via an opsonization-dependent mechanism and binds apoptotic PMNs approximately 4-fold more than viable PMNs. Also, binding of SP-A to apoptotic PMNs does not appear to involve SP-A's lectin domain. These data suggest that the pulmonary collectins SP-A and SP-D facilitate the resolution of inflammation by accelerating apoptotic PMN clearance.
Pentraxins are acute-phase proteins produced in vivo during inflammatory reactions. Classical short pentraxins, C-reactive protein, and serum amyloid P component are generated in the liver in response to interleukin (IL)–6. The long pentraxin PTX3 is produced in tissues under the control of primary proinflammatory signals, such as lipopolysaccharide, IL-1β, and tumor necrosis factor-α, which also promote maturation of dendritic cells (DCs). Cell death commonly occurs during inflammatory reactions. In this study, it is shown that PTX3 specifically binds to dying cells. The binding was dose dependent and saturable. Recognition was restricted to extranuclear membrane domains and to a chronological window after UV irradiation or after CD95 cross-linking–induced or spontaneous cell death in vitro. PTX3 bound to necrotic cells to a lesser extent. Human DCs failed to internalize dying cells in the presence of PTX3, while they took up normally soluble or inert particulate substrates. These results suggest that PTX3 sequesters cell remnants from antigen-presenting cells, possibly contributing to preventing the onset of autoimmune reactions in inflamed tissues.
Immune context is an essential determinant of the host response to potential autoantigens. The clustering of the autoantigens targeted in systemic lupus erythematosus within surface blebs of apoptotic cells generates high concentrations of autoantigen within discrete subcellular packages. We demonstrate here that when apoptosis is induced by Sindbis virus infection, viral antigens and autoantigens cocluster exclusively in small surface blebs of apoptotic cells. The surface of these blebs is rich in viral glycoproteins, and virions can be seen blebbing from their surface. We propose that these blebs of mixed foreign and self-origin define a novel immune context that may challenge self-tolerance.
Objective
To investigate whether the established impaired phagocyte function in systemic lupus erythematosus (SLE) patients also affects apoptotic cell clearance. Accumulation of apoptotic waste as a source for autoantigens that induce and maintain autoimmune responses is discussed.Methods
Apoptosis was detected by morphology and propidium iodide staining. In vitro phagocytosis of autologous apoptotic cells in cultured peripheral blood mononuclear cells was evaluated microscopically. Cross-feeding experiments were performed to investigate phagocytosis of heterologous apoptotic cells by in vitro-differentiated macrophages. Furthermore, the effect of annexin V on the phagocytosis of apoptotic cells was investigated.ResultsReduced clearance of apoptotic cells in SLE patients was observed. The defective clearance appeared to reflect phagocyte dysfunction and not an abnormal execution of apoptosis. A similar picture was seen when in vitro-differentiated macrophages from control populations were treated with annexin V.Conclusion
Noninflammatory engulfment phagocytosis of apoptotic cells is decreased in SLE patients. Persistently circulating apoptotic waste may encounter inflammatory removal pathways and serve as immunogen for the induction of autoreactive lymphocytes and as antigen for immune complex formation.
The 1st International Workshop on Nucleotides and their Receptors in the Immune System was held on 8–10 September 2000 at the University of Ferrara, Ferrara, Italy.
To determine the relative contributions of Fc- and complement-mediated immune clearance to the overall mononuclear phagocyte system (MPS) dysfunction in SLE, we performed a kinetic analysis of clearance rate data from 32 patients and 49 normal controls. Three rate constants regulating complement-mediated MPS clearance and one rate constant regulating Fc-mediated MPS clearance were evaluated. Mean values for rate constants regulating complement-mediated phagocytosis (k4) and Fc-mediated clearance (k3) were significantly lower for the patient population as a whole when compared with normal controls (p less than 0.01 and p less than 0.001, respectively). Further analysis by clinical subgroups revealed that mean k3 values were significantly low for all but the inactive nonrenal subset of patients, whereas mean k4 values were significantly low for both the active and inactive renal patients but not for nonrenal patients. Rate constant values for Fc-mediated clearance correlated significantly with disease activity scores for the entire SLE group (p less than 0.001) as well as for the subset of patients with active and inactive renal disease (p less than 0.001). Neither k3 nor k4 correlated significantly with anti-DNA antibody, titers, total hemolytic complement levels, or circulating immune complexes. These data indicate that both Fc- and complement-mediated clearance defects occur in SLE. Nonrenal patients have at least one clearance mechanism intact, whereas immune complex glomerulonephritis is associated with dysfunctions in both of these clearance mechanisms.
Cell death takes two distinct forms, necrosis and apoptosis. Necrosis is a degenerative phenomenon that follows irreversible injury. Apoptosis, in contrast, appears to be an active process requiring protein synthesis for its execution; it is implicated in physiological regulation of tissue size, and, where it occurs pathologically, a homeostatic role for the death is often evident. Morphologically, apoptosis involves condensation of the nuclear chromatin and cytoplasm, fragmentation of the nucleus, and budding of the whole cell to produce membrane-bounded bodies in which organelles are initially intact. These bodies are disposed of by adjacent cells without inflammation. Biochemically, there is distinctive internucleosome cleavage of DNA in apoptosis, which is quite different from the random DNA degradation observed in necrosis.
Follicular dendritic cells (FDC), isolated from human tonsils or adenoids, were tested for their capacity to retain monomeric, aggregated or antigen-bound human antibodies in the absence of serum. FDC retain fluorescein-labelled heat-aggregated human immunoglobulins, but not monomeric ones nor fluorescein-labelled F(ab')2 in monomeric or aggregated form. Ultrastructural observations showed that colloidal gold-labelled monomeric, or antigen-bound, antibodies directed against tetanus toxoid are retained by dendrites and membrane infoldings of FDC but are never located in cytoplasmic vesicles. This retention was inhibited by incubating FDC with unlabelled aggregated or antigen-bound antibodies. When gold-labelled anti-tetanus toxoid antibodies were incubated in the presence of protein-A before the contact with FDC, a strong reduction of their retention occurred. This further suggested the presence of Fc receptors on isolated tonsillar FDC. Endocytosis was not observed in isolated FDC, even after prolonged incubation in presence of labelled immune complexes: their Fc receptors are, thus, not related to a phagocytic activity as they are in macrophages. Simultaneous ultrastructural labelling of Fc and C3b receptors with colloidal gold particles of different sizes did not reveal any clear relations between these two receptors on the surface of FDC.
Serum deoxyribonuclease I (DNase I) activity in systemic lupus erythematosus (SLE) patients was shown to be lower than that of healthy laboratory personnel, rheumatoid arthritis, and scleroderma patients (P less than 0.001). The decrease in DNase I activity in SLE sera was not due to the effect of various autoantibodies or to heat labile DNase I inhibitor. A relationship between serum DNase I activity and active SLE was demonstrated. Patients with active lupus nephritis had the lowest levels of enzymatic activity.
Fc receptor-mediated mononuclear phagocyte system (MPS) clearance is impaired in systemic lupus erythematosus (SLE) and may contribute to the pathogenesis of the immune complex disease. To investigate the basis of MPS dysfunction, we have examined concurrent in vivo and in vitro Fc receptor function in 22 patients with SLE and 23 disease-free adults. Blood monocyte Fc receptor binding was increased rather than decreased as predicted by the saturation hypothesis of MPS blockade. Rosette formation of IgG-sensitized bovine erythrocytes (EA) with monocytes demonstrated increased Fc receptor-ligand binding in SLE (percent rosettes: 40 +/- 12 vs 27 +/- 8, p less than 0.001). Scatchard analysis of the binding of radiolabeled IgG oligomers to SLE monocytes indicated a mean receptor number 30% higher than control, although this did not reach statistical significance. Despite enhanced Fc receptor-ligand (EA) binding, Fc-mediated phagocytosis of EA was decreased in SLE (1.7 +/- 0.7 erythrocytes/monocytes/hour vs 2.6 +/- 1.0, p less than 0.004). This decrease in phagocytosis by blood monocytes from SLE patients was significantly greater than that attributable to the predominance in SLE of individuals with certain HLA B cell alloantigens and intrinsically lower phagocytic rates (p less than 0.05 for all groups). This decrease therefore represents a disease-acquired characteristic. Furthermore, the phagocytic rate of the four SLE patients with marked prolongation in MPS clearance was significantly lower than that of the eight patients with near normal clearance values (p less than 0.01). Saturation of Fc receptors by immune complexes does not explain impaired immune clearance in SLE. Our results indicate that despite increased binding of the EA ligand, Fc receptor-mediated phagocytosis is markedly impaired in SLE monocytes. This impairment cannot be explained on the basis of HLA-related differences in phagocytosis among lupus patients. The defect in phagocytosis of EA is most profound in those patients with the most significantly impaired MPS clearance. Thus, the dissociation of receptor-ligand binding and receptor-mediated internalization may contribute significantly to the in vivo clearance defect in SLE.
Extensive studies in murine models of lupus nephritis have shown that cationic anti-DNA autoantibodies have nephritogenic potential. We have investigated whether cationic anti-DNA antibodies of IgG class are also produced in vivo in patients with active lupus nephritis. Antibodies against DNA in the sera from patients with SLE were purified by affinity chromatography on DNA-cellulose, followed by nonequilibrium pH gradient electrophoresis and SDS-PAGE. The highly cationic anti-DNA antibodies of IgG class were prominent in the nonequilibrium pH gradient electrophoresis-immunoblots of the antibodies from the patients with active lupus nephritis. Decreased proteinuria after successful treatment with prednisolone was associated with disappearance of the cationic anti-DNA antibodies in the circulation. The cationic anti-DNA antibodies did bind to heparan sulfate, which is a major glycosaminoglycan in glomerular basement membrane, much better than did neutral anti-DNA antibodies. The results suggest that the cationic anti-DNA autoantibodies may play a certain role in the development of lupus nephritis. Our study demonstrates the strong association between the presence of cationic anti-DNA antibody and the development of lupus nephritis in humans.
Plasma and whole-body turnover studies of human C-reactive protein (CRP), isolated from a single normal healthy donor and labeled with 125I, were undertaken in 8 healthy control subjects and 35 hospitalized patients including cases of rheumatoid arthritis, systemic lupus erythematosus, infections, and neoplasia. Plasma clearance of 125I-CRP closely approximated to a monoexponential function and was similar in the control and all patient groups. There was no evidence for accelerated clearance or catabolism of CRP in any of the diseases studied. The 19-h half-life was more rapid than that of most human plasma proteins studied previously, and the fractional catabolic rate was independent of the plasma CRP concentration. The synthesis rate of CRP is thus the only significant determinant of its plasma level, confirming the validity of serum CRP measurement as an objective index of disease activity in disorders associated with an acute-phase response. Approximately 90% of injected radioactivity was recovered in the urine after 7 d, and scintigraphic imaging studies with 123I-labeled CRP in 10 patients with different focal pathology showed no significant localization of tracer. The functions of CRP are thus likely to be effected predominantly in the fluid phase rather than by major deposition at sites of tissue damage or inflammation.
Cells can die by two distinct pathways: apoptosis or necrosis. Necrosis is associated with rapid metabolic collapse that leads to cell swelling, early loss of plasma membrane integrity, and ultimate cell rupture. Cytosolic contents leak from the necrotic cell causing injury and inflammation to surrounding tissue. In contrast, apoptosis is an energy-requiring, gene-directed process, which, when activated, results in cell "suicide." The morphological and biochemical characteristics of cells dying by apoptosis differ markedly from those of cells dying by necrosis. During apoptosis, cells decrease in size and round up. The nuclear chromatin undergoes condensation and fragmentation. The apoptotic cell then breaks apart into many plasma membrane-bound vesicles called "apoptotic bodies," which contain fragments of condensed chromatin and morphologically intact organelles such as mitochondria. Apoptotic cells and bodies are rapidly phagocytosed, thereby protecting surrounding tissues from injury. The rapid and efficient clearance of apoptotic cells makes apoptosis extremely difficult to detect in tissue sections. Recent studies show that multiple cytotoxic stimuli well known to cause necrosis can lead to apoptosis instead when cells are exposed to the same noxious agents at lower concentrations. This insight has led to an interest in the role of apoptosis in the pathogenesis of renal diseases that result primarily from injury to renal tubular epithelial cells. These diseases include acute and chronic renal failure from exposure of the kidney to ischemia or to cytotoxic agents. In this review we discuss some relevant aspects of the differences between necrotic and apoptotic cell death. We also present evidence to support the hypothesis that apoptosis is an important pathogenic mechanism in those forms of acute and chronic renal failure in which the renal tubular epithelial cell is the primary target of ischemic or toxic injury.
To analyze factors related to apoptosis in systemic lupus erythematosus (SLE) peripheral blood mononuclear cells (PBMC) and to compare the findings in SLE PBMC with those in normal donor PBMC or PBMC from patients with other autoimmune diseases.
PBMC from normal healthy donors or patients with SLE, mixed connective tissue disease (MCTD), rheumatoid arthritis (RA), or various vasculitides were isolated. The percentage of apoptosis after activation through different signaling pathways was quantified using propidium iodide staining. Protein expression of Fas/APO-1 or bcl-2, and messenger RNA (mRNA) expression of bcl-2, bcl-xL, bax, bak, Fas/APO-1, Fas ligand (Fas-L), c-myc, mad, or max were determined.
We confirmed previous findings of increased numbers of apoptotic cells in SLE PBMC compared with normal donor cells after in vitro incubation. After activation of PBMC with CD28 monoclonal antibody plus phorbol myristate acetate (CD28 MAb/ PMA), staphylococcal enterotoxin B (SEB), or phytohemagglutinin (PHA), the percentage of apoptotic cells was unchanged (SEB) or diminished (CD28 MAb/PMA, PHA) in SLE cells, and the difference between normal donor and SLE cells was less pronounced. On the mRNA level, expression of apoptosis-related gene products did not differ between SLE cells and normal donor cells. Expression of Fas/APO-1 protein was increased in freshly isolated SLE T lymphocytes compared with normal donor T lymphocytes, whereas bcl-2 protein was up-regulated after a 3-day culture period. Cellular activation further increased bcl-2 protein levels, eliminating differences between normal donors and SLE patients. In RA cells, the percentage of apoptosis was similar to that in normal donor PBMC, whereas results using cells from patients with other autoimmune diseases (MCTD, Wegener's granulomatosis, Takayasu arteritis, polyarteritis nodosa) were comparable with those found using SLE PBMC. Addition of growth factors such as interleukin-2 (IL-2), IL-4, or IL-15 to culture medium decreased the percentage of in vitro apoptosis in both normal donor and SLE cells.
Based on these data, we conclude that accelerated in vitro apoptosis and increased Fas/ APO-1 and bcl-2 protein expression in SLE are nonspecific for the disease, and might be explained at least in part by the increased in vivo activation levels of PBMC from patients with SLE, MCTD, or autoimmune vasculitides combined with in vitro incubation under "noninflammatory" conditions and growth factor withdrawal.
Complete deficiency of C1q is almost invariably associated with the development of systemic lupus erythematosus. It has been suggested that this association may result from a generalized failure to clear Ag-Ab complexes. However, it has not been demonstrated how such a broad impairment results in this specific and consistent autoimmune phenotype, in which photosensitive skin disease is the most prominent manifestation. We believe there is another role for the classical pathway in maintaining immune tolerance. Surface blebs of apoptotic keratinocytes are concentrated sources of autoantigens, and these packages may define a novel immune context and challenge self-tolerance if not properly cleared and processed. We demonstrate here that when human keratinocytes are rendered apoptotic, they also develop the capacity to specifically and directly bind to C1q in the absence of Ab. C1q may mediate Ab-independent clearance of apoptotic keratinocytes, and prevent immunization with autoantigens of cutaneous origin.
A new radial enzyme diffusion (RED) method for the measurement of DNase activity in serum and urine is described. The sensitivity of the assay is in the range of 15.6-500 ng/ml. The assay is based on the hydrolysis of double-stranded (ds) DNA (or nucleosomes) in agarose. The specificity of the reaction for DNase I was established by showing that either EDTA in the reaction buffer or G-actin abolished DNase activity. Being a functional assay, RED has advantages over radioimmunoassay (RIA) or ELISA, since antigenic assays may also measure complexes of DNase with actin. This method was used to measure DNase activity in the sera and urine of lupus-prone mice (NZB/NZW F1 hybrids, aged 4-6 weeks). Serum DNase activity in these mice was significantly lower (mean 9 ng/ml) than in control, normal mice of the same age and sex (mean 37 ng/ml). Concentration of DNase in the urine of 4-6-week-old female NZB/NZW F1 hybrids (24 ng/ml) was significantly lower then in control mice (521 ng/ml). The RED method was used to measure the concentration of actin as the DNase inhibitor in serum. G-actin in the presence of ATP binds DNase and inhibits its nucleolytic activity. Since ATP is necessary for the actin inhibition of DNase I, this shows that there is actin as well as DNase I in the serum. Actin is not only ATP-dependent, but also heat-labile. Heating the sera for 10 min at 50 degrees C increases DNase activity. This is an alternative method for measuring the concentration of actin in the serum. An almost identical estimate of actin concentration in sera of normal mice was found from the difference of DNase activity in the presence or absence of ATP (mean actin concentration = 21 ng/ml) or from the difference of DNase activity in heated and non-heated serum (mean actin concentration 18 ng/ml). We were not able to demonstrate DNase inhibitors in the urine of either control or NZB/W F1 hybrid mice.
In vivo, the normal fate of cells undergoing apoptosis is recognition, uptake and degradation of the intact dying cell by phagocytes.
Cell clearance by this mechanism is fast, efficient and injury-limiting, being mediated by macrophages and semi-professional
phagocytes. Apoptotic cells are marked for disposal by mechanisms which remain poorly understood, although in some circumstances
surface sugar changes and exposure of phosphatidylserine lead to recognition by uncharacterised phagocyte receptors. Furthermore,
there is specific evidence in vitro for involvement of phagocyte receptors including the thrombospondin receptors αβ3 and CD36, scavenger receptors, the 61D3 antigen and the ABC1 transporter. It is conceivable that recognition mechanisms may
be ordered in a hierarcht of ‘back ups’, each recognising cells at different stages of the death program. Nevertheless, a
full understanding of this complexity will require definition of recognition mechanisms which operate in vivo in higher organisms.
Increased in vitro apoptosis and altered expression of apoptosis-related molecules have been reported in systemic lupus erythematosus (SLE). It was speculated that autoantigens released from apoptizing cells may contribute to the etiopathogenesis of SLE by both activation of autoreactive lymphocytes and the formation of immune complexes. Conflicting data about the level of cellular apoptosis from animal models for SLE and human SLE indicate different pathomechanisms.
In the present study we analysed the count of circulating early apoptotic cells and its correlation with the clinical activity in SLE compared with the amount in primary systemic vasculitis (PSV). The percentage of early apoptotic cells determined by Annexin V-FITC binding cells was significantly increased in peripheral blood of SLE patients compared with normal healthy donors (NHD) and PSV (NHD 5.62%, SLE 13.68%, PSV 8.69%). Analysis of lymphocytes revealed significantly increased counts in the T cell populations (NHD 5.15%, SLE 12.22%, PSV 10.01%), whereas the increase in B cells did not reach significance level (NHD 9.20%, SLE 18.95%, PSV 16.59%). Apoptotic cell count revealed no correlation to SLAM-Score or to immunosuppressive therapy, corticosteroid-dosage or absolute lymphocyte count.
The general increase of circulating apoptotic cells may indicate an impaired clearance in SLE patients, independent of clinical activity or therapeutic interventions. Autoantigens from apoptotic cells may contribute to both activation of autoreactive lymphocytes and formation of immune complexes.
Much of the attention on protein antigens and their induction of T-cell responses has focused on the nature of the interactions of their `core' determinants with the major histocompatibility complex (MHC) or the T-cell receptor (TCR). Here, Kamal Moudgil and colleagues instead emphasize the vital role of the amino acid residues that flank the core antigenic determinant on the outcome of the response.
Autoantibodies to CRP were reported previously in patients suffering from toxic oil syndrome. This syndrome resembles autoimmune diseases such as systemic lupus erythematosus (SLE) or systemic scleroderma. We therefore examined the prevalence of antibodies to CRP and other acute-phase proteins in autoimmune diseases, including SLE, subacute cutaneous lupus erythematosus (SCLE), systemic scleroderma (SSc), and primary biliary cirrhosis (PBC), as well as in bone marrow transplantation-induced chronic graft-versus-host disease and eosinophilia-myalgia syndrome. IgG antibodies to CRP were found in 78% of SLE and in 30% of SCLE patients, while 16% of patients with PBC were positive. In up to 45% of patients with SSc predominantly IgG antibodies to ceruloplasmin were detectable. Lack of systemic involvement as in discoid lupus erythematosus and localized scleroderma (morphea) correlated with low or absent antibody formation. However, no correlation was found between anti-acute-phase protein antibodies with liver disease or other organ involvement. Adsorption studies revealed that non-native epitopes on the CRP molecule, termed modified CRP, are the main target of antibodies. Chronic inflammatory tissue injury in systemic autoimmune disease might increase the presentation of cryptic epitopes of CRP to the threshold required for T cell activation.
Systemic autoimmune diseases are a genetically complex, heterogeneous group of diseases in which the immune system targets a diverse, but highly specific group of intracellular autoantigens. The clustering and marked concentration of these molecules in the surface blebs of apoptotic cells, and their modification by apoptosis-specific proteolytic cleavage and/or phosphorylation at these sites, has focused attention on a unique apoptotic setting as the potential initiating stimulus for systemic autoimmunity. This apoptotic event is likely to (i) occur in a microenvironment containing high concentrations of the targeted antigens, (ii) be pro-immune in nature (e.g. viral infection), and (iii) allow suprathreshold concentrations of antigen with non-tolerized structure (either novel fragments, post-translational modifications, or complexes) to enter the class II processing pathway and initiate a primary immune response. Defective clearance or reduced anti-inflammatory consequences of apoptotic material may be important susceptibility factors in this group of diseases. Once the primary immune response to apoptotic antigens has been initiated, other apoptotic events (occurring in the course of homeostasis or damage) may stimulate the secondary immune response with less stringency, resulting in flares.
The cardinal feature of systemic lupus erythematosus is the formation of anti-nuclear antibodies. In recent years, it has become clear that the nucleosome is a major autoantigen that drives this T cell-dependent autoimmune response, as exemplified by the presence of nucleosome-specific T helper cells and the high prevalence of nucleosome-specific autoantibodies. The only way to generate nucleosomes in vivo is by the process of apoptosis. There is growing evidence that in systemic lupus erythematosus apoptosis is disturbed, leading to the release of nucleosomes. Moreover, apoptosis-induced modifications of these autoantigens may render them more immunogenic, especially if the removal of apoptotic cells is insufficient. The first indications for the impaired clearance of apoptotic cells in systemic lupus erythematosus are emerging. Nucleosomes are also important for mediating tissue lesions, especially glomerulonephritis. In lupus nephritis nucleosomes, nucleosome-specific antibodies and nucleosome/IgG complexes have been identified in the glomerular immune deposits. Via their cationic histone part nucleosomes mediate the binding of anti-nuclear antibodies to intrinsic constituents of the glomerular basement membrane, such as the anionic heparan sulfate and collagen IV. Appreciation of this binding mechanism may lead to new treatment strategies, as shown for non-coagulant heparinoids.
The T cell repertoire is shaped by the processes of positive and negative selection. We have previously shown that mice are tolerant to a native self-Ag, mouse lysozyme (ML), but they respond vigorously when challenged with different ML peptides ("cryptic" self-determinants). In this study, we have addressed the issue of the physiological significance of both the hierarchy (dominance/crypticity) of self-determinants within ML and the anti-cryptic, self (ML)-directed T cell repertoire. Our results demonstrate that there are several ML peptides that bind well to MHC but are totally nonimmunogenic when tested for proliferative T cell response and cytokine secretion: a subset of these peptides presumably represent the originally dominant self-determinants of ML, which have rendered the T cells tolerant during thymic selection. Other ML peptides, which bind well to MHC and are immunogenic, correspond to the cryptic determinants of ML: T cells against cryptic ML determinants escape tolerance induction. Thus, the mature T cell repertoire against ML bears the direct imprint of the hierarchy of self (ML)-determinants. Interestingly, hen egg white lysozyme could prime T cells in vivo that were cross-reactive with certain cryptic ML determinants, and vice versa, without requiring any coimmunization with the foreign lysozyme and ML peptide(s). Moreover, repeated, deliberate priming and expansion of T cells by hen egg white lysozyme immunization concomitantly enhanced T cell response to such cross-reactive ML determinants. This reciprocal self-foreign determinant cross-reactivity may play a previously unrecognized, but crucial, role in the expansion and diversification of self-reactive clones in the autoimmune response.
cytosis of cellular corpses. During apoptosis, the asymmetry of plasma membrane phospholipids is lost, which exposes phosphatidylserine externally. The phagocytosis of apoptotic cells can be inhibited stereospecifically by phosphatidylserine and its structural analogues, but not by other anionic phospholipids, suggesting that phosphatidylserine is specifically recognized. Using phage display, we have cloned a gene that appears to recognize phosphatidylserine on apoptotic cells. Here we show that this gene, when transfected into B and T lymphocytes, enables them to recognize and engulf apoptotic cells in a phosphatidylserine-specific manner. Flow cytometric analysis using a monoclonal antibody suggested that the protein is expressed on the surface of macrophages, fibroblasts and epithelial cells; this antibody, like phosphatidylserine liposomes, inhibited the phagocytosis of apoptotic cells and, in macrophages, induced an anti-inflammatory state. This candidate phosphatidylserine receptor is highly homologous to genes of unknown function in Caenorhabditis elegans and Drosophila melanogaster, suggesting that phosphatidylserine recognition on apoptotic cells during their removal by phagocytes is highly conserved throughout phylogeny.
Apoptotic and necrotic cells are strong candidates as sources of the autoantigens that drive the autoantibody response in systemic lupus erythematosus (SLE). Defects in the physiological mechanisms for the clearance of dying cells may promote disease susceptibility to SLE. Ablation of the mouse gene Dnase1 results in the development of anti-chromatin autoimmunity and glomerulonephritis, indicating that the enzyme protects against autoimmunity by digesting extracellular chromatin.