Mechanisms of Chromatin Remodeling and Repurposing During Extracellular Translocation.

Chromatin is a highly conserved molecular structure that provides genetic information to regulate cell function. Comprised of DNA, histones and interacting proteins, chromatin is inherently dynamic and subject to remodeling. While usually conceptualized as an intranuclear event, remodeling can also involve extracellular movement. Indeed, chromatin can translocate entirely from the inside to the outside of the cell during cell death processes that include apoptosis, necrosis, and NETosis. During these processes, DNA and proteins can undergo other changes impacting on their activity. Thus, during apoptosis, DNA can be cleaved, histones can be posttranslationally modified and a nuclear protein called HMGB1 (high mobility group box 1) can undergo redox changes. Outside the cell, chromatin components can display powerful immunological activities. These activities result from the ability of DNA and RNA, once taken up by immune cells, to activate internal nucleic acid sensors; the likely function of these sensors is to recognize nucleic acids from intracellular infection. Depending on redox state, the prototype alarmin HMGB1 can interact with a variety of immune receptors including Toll-like receptors. As such, extracellular chromatin can stimulate inflammation and drive the pathogenesis of immune-mediated diseases; in experimental models in animals, agents that bind chromatin components can block disease. Thus, extracellular chromatin can have far-reaching biological effects involving a form of molecular repurposing.

The Role of Cell Death in the Pathogenesis of SLE: Is Pyroptosis the Missing Link?

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of antinuclear antibodies (ANAs) in association with systemic inflammation and organ damage. In addition to genetic factors, a contribution of infection to disease induction has been proposed. In the pathogenesis of lupus, immune complexes of ANAs with nuclear antigens can form and both deposit in the tissue and stimulate cytokine production to intensify inflammation. As such, the extracellular release of nuclear antigens to form pathogenic immune complexes is an important step in the pathway to disease. This release has been considered the consequence of cell death, with apoptotic cells the relevant source of nuclear material. While apoptosis could serve this role, other death forms may act similarly. Among these death forms, pyroptosis, which can be induced by inflammasome activation during infection, has features suggesting involvement in lupus. Thus, unlike apoptosis, pyroptosis is a pro-inflammatory process. Furthermore, pyroptosis leads to the release of intracellular contents including HMGB1 and ATP, both of which can act as DAMPs (death associated molecular patterns) to stimulate further inflammation. Importantly, pyroptosis can lead to the release of intact nuclei, suggesting a relationship to events in the formation of LE cells. While the role of pyroptosis in SLE is hypothetical at this time, further analysis of this death form should provide new insights into lupus pathogenesis and provide the missing link between infection and the initiation of lupus by products of dead and dying cells.

The expression of cytokines and chemokines in the blood of patients with severe weight loss from anorexia nervosa: an exploratory study.

Anorexia nervosa (AN) is a serious, potentially life-threatening disorder characterized by severe weight loss, dysregulated eating, and often excessive exercise. While psychiatric illnesses such as depression are associated with increased levels of pro-inflammatory mediators, evidence for such disturbances in patients with AN has been less clear. In an exploratory study of possible disturbances in immune responses in AN, we assayed a panel of cytokines and chemokines in the blood of patients undergoing inpatient treatment, testing the hypothesis that metabolic disturbances in this disease would lead to a pattern of immune disturbances distinct from that of other psychiatric diseases. For this purpose, we evaluated patients by the Beck Depression Inventory-II (BDI-II) and the Eating Disorders Examination-Questionnaire and assessed cytokines and chemokines by enzyme-linked immunosorbent assays. Patients reported a moderate level of depression (mean BDI-II = 22.6) but exhibited few immunologic abnormalities of the kind associated with major depressive disorder [e.g., increased interleukin (IL)-6]; RANTES showed the most frequent elevations and was increased in 4 of the patients studied. Together, these findings suggest that features of AN such as loss of adipose tissue and excessive exercise may attenuate cytokine production and thus modulate the experience of illness that impacts on core features of disease.

The expression of HMGB1 on microparticles from Jurkat and HL-60 cells undergoing apoptosis in vitro.

HMGB1 is a highly conserved nuclear protein that displays important biological activities inside as well as outside the cell and serves as a prototypic alarmin to activate innate immunity. The translocation of HMGB1 from inside to outside the cell occurs with cell activation as well as cell death, including apoptosis. Apoptosis is also a setting for the release of cellular microparticles (MPs), which are small membrane-bound vesicles that represent an important source of extracellular nuclear molecules. To investigate whether HMGB1 released from cells during apoptosis is also present on MPs, we determined the presence of HMGB1 on particles released from Jurkat and HL-60 cells induced to undergo apoptosis in vitro by treatment with either etoposide or staurosporine; MPs released from cells undergoing necrosis by freeze-thaw were also characterized. As shown by both Western blot analysis and flow cytometry, MPs from apoptotic cells contain HMGB1, with binding by antibodies indicating an accessible location in the particle structure. These results indicate that HMGB1, like other nuclear molecules, can translocate into MPs during apoptosis and demonstrate another biochemical form of this molecule that may be immunologically active.

The role of microparticles in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus.

Microparticles (MPs) are small membrane-bound vesicles with potent biological activities that can promote the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus (SLE). These particles contain diverse cellular components and are shed from cells during apoptosis or activation. MPs can drive inflammation and autoimmunity by multiple mechanisms reflecting their content of bioactive molecules and ability to engage multiple receptor systems simultaneously. In the rheumatoid joint, particles can stimulate synovitis via their display of cytokines, chemokines, complement and angiogenesis factors. In SLE, particles can serve as an important source of extracellular nuclear molecules to signal 'danger' and form pathogenic immune complexes. Future studies will define the pathways by which particles promote pathogenesis, strategies to block their activity and their utility as biomarkers to assess disease activity and the response to therapy.

Effect of lipopolysaccharide administration on the number, phenotype and content of nuclear molecules in blood microparticles of normal human subjects.

Microparticles (MPs) are small membrane-bound vesicles that arise from activated and dying cells and promote inflammation and thrombosis. To characterize the in vivo release of MPs, we used flow cytometry to measure MPs in the blood of 15 healthy volunteers administered bacterial endotoxin (lipopolysaccharide or LPS) in the presence of a low dose of hydrocortisone with or without inhaled nitric oxide. MPs, defined as particles less than 1.0 µm in size, were assessed following labelling for CD42a, CD14 and CD62E or CD144 antibodies to identify MPs from platelets (PMPs), monocytes (MMPs) and endothelial cells (EMPs). In addition, PMPs and MMPs were labelled with anti-HMGB1 and stained with SYTO13 to assess nuclear acid content. Administration of LPS led to an increase in the numbers of PMPs, MMPs and EMPs as defined by CD62E, as well as the number of MMPs and PMPs staining with anti-HMGB1 and SYTO13. Inhalation of NO did not influence these findings. Together, these studies show that LPS can increase levels of blood MPs and influence phenotype, including nuclear content. As such, particles may be a source of HMGB1 and other nuclear molecules in the blood during inflammation.

The effects of heparins on the liver: application of mechanistic serum biomarkers in a randomized study in healthy volunteers.

Heparins have been reported to cause elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) but have not been associated with clinically significant liver injury. The mechanisms underlying these benign laboratory abnormalities are unknown. Forty-eight healthy men were randomized to receive subcutaneous injections of unfractionated heparin (UFH; 150 U/kg), enoxaparin sodium (1 mg/kg), dalteparin sodium (120 IU/kg), or adomiparin sodium (125 IU/kg; a novel heparin) every 12 h for 4.5 days. Asymptomatic elevations in serum ALT or AST were observed in >90% of the subjects. Elevations were also observed in the levels of serum sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GLDH), miR-122, high-mobility group box-1 protein (including the acetylated form), full-length keratin 18, and DNA. Keratin 18 fragments, which are apoptosis biomarkers, were not detected. Biomarker profiles did not differ significantly across heparin treatments. We conclude that heparins as a class cause self-limited and mild hepatocyte necrosis with secondary activation of an innate immune response.

Antinuclear antibodies in rheumatic disease: a proposal for a function-based classification.

Antinuclear antibodies (ANAs) are a diverse group of autoantibodies that bind macromolecular components of the cell nucleus. While some ANAs occur in normal individuals, others are expressed almost exclusively in patients with rheumatic disease and serve as markers for diagnosis and prognosis. Despite the clinical associations of ANAs, the relationship of these antibodies to specific disease manifestations is often unknown because the target antigens are intracellular molecules that are ubiquitously expressed. In systemic lupus erythematosus, the role of ANAs in disease manifestations is better understood, especially for antibodies to DNA and related nucleosomal antigens. These antibodies can promote nephritis by the formation of immune complexes that are deposited in the kidney. In addition, anti-DNA, along with antibodies to RNA-binding proteins such as anti-Sm, can induce non-specific immune abnormalities based on the induction of type interferon 1 by plasmacytoid dendritic cells. Despite ANA expression in rheumatic disease, studies in animal models of inflammation and tissue injury indicate that antibodies to certain nuclear molecules such as HMGB1 have protective effects. Together, these considerations suggest a function-based classification of ANAs based on their expression in normal and autoimmune individuals as well as their capacity to induce or attenuate immunological disturbances. This classification provides a framework to elucidate the serological features of rheumatic disease and the often uncertain relationship between ANA expression and disease manifestations.

Antibodies to DNA: infection or genetics?

Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE) and unique markers of the immunological disturbances critical to disease pathogenesis. In the form of immune complexes, anti-DNA autoantibodies can deposit in the tissue to incite inflammation and damage; in addition, these complexes can induce cytokine production, most prominently, type 1 interferon. Studies in both patients and animal models have implicated genetic as well as environmental factors in the aetiology of the anti-DNA response. Because bacterial DNA is a potent stimulant of innate immunity by both toll-like receptor (TLR) and non-TLR signalling pathways, foreign DNA introduced during the course of bacterial or viral infection could have a dual role in antibody induction. This DNA could serve as an adjuvant to activate innate immunity as well as an immunogen to drive an antigen-specific antibody response. In this scenario, the generation of cross-reactive autoantibodies, in contrast to highly specific antibodies to bacterial DNA, most likely depends on genetically determined abnormalities in the B-cell repertoire in patients with SLE. Given the universal expression of DNA, this model suggests that many different kinds of infections could trigger pathogenic autoantibody responses in SLE, as well as induce flare.

The role of extracellular DNA in autoimmunity in SLE.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the expression of antibodies to DNA. These antibodies form immune complexes that can stimulate cytokine production as well as deposit in the tissues to incite inflammation and damage. For the formation of immune complexes, the availability of extracellular DNA in an immunologically relevant form is essential. While apoptosis has been implicated as the source of this nuclear material in SLE, as shown with in vitro or in vivo systems, extracellular DNA can originate from apoptotic as well as necrotic cells. In experimental models, the release of DNA occurs with the administration of cells induced to die, in vitro as well as the administration of agents to induce cell death in situ. This release can be influenced by the presence of inflammatory cells such as macrophages that can interact with dead and dying cells to modulate the translocation of DNA from the inside to the outside of cells. In vivo, both glucocorticoids and oestrogens can modify the extent of DNA release from the administration of dead and dying cells. Together, these findings indicate that the generation of extracellular DNA in SLE can result from cell death and that steps in this process represent potential targets for new therapies.

The role of microparticles in inflammation and thrombosis.

Microparticles (MP) are small membrane-bound vesicles that circulate in the peripheral blood and play active roles in thrombosis, inflammation and vascular reactivity. While MP can be released from nearly every cell type, most investigation has focused on MP of platelet, leucocyte and endothelial cell origin. Cells can release MP during activation or death. Flow cytometry is the usual method to quantify MP; the small size of these structures and lack of standardization in methodology complicate measurement. As MP contain surface and cytoplasmic contents of the parent cells and bear phosphatidylserine, antibodies to specific cell surface markers and annexin V can be used for identification. Through various mechanisms, MP participate in haemostasis and have procoagulant potential in disease. MP contribute to inflammation via their influence on cell-cell interactions and cytokine release, and MP also function in mediating vascular tone. In several disease states characterized by inflammation and vascular dysfunction, MP subpopulations are elevated, correlate with clinical events, and may have important roles in pathogenesis. In the rheumatic conditions such as rheumatoid arthritis and systemic lupus erythematosus, MP are potentially important markers of disease activity and have an increasingly recognized role in immunopathogenesis. It is clear that MP play an important role in atherosclerosis, and study of these structures may provide insight into the link between chronic inflammatory conditions and accelerated atherosclerosis. As biomarkers, MP allow access to usually inaccessible tissues such as the endothelium. Further research will hopefully lead to interventions targeting MP release and function.

The generation of extracellular DNA in SLE: the role of death and sex.

DNA is a large macromolecule that plays a central role in the pathogenesis of systemic lupus erythematosus (SLE), serving as a target antigen of autoantibodies as well as a major component of immune complexes. These complexes can both promote immune disturbances as well as deposit in the kidney to incite inflammation. While the origin of anti-DNA autoantibodies in SLE has received intense investigation, the mechanisms by which DNA exits cells to form immune complexes in the circulation is not well understood. To determine the origin of DNA circulating in the blood in SLE, our laboratory has been using a murine model system to track the in vivo fate of DNA from Jurkat T cells that have been made apoptotic or necrotic in vitro and then administered to mice. Results of these studies indicate that DNA from apoptotic and necrotic cells appears in the blood in a time- and dose-dependent manner. Irrespective of origin, this DNA has properties of nucleosomes as shown by its molecular weight. The process of release requires the presence of macrophages and can be modified by glucocorticoids as well as inflammation. In addition, sex may play a role in the generation of extracellular DNA from dead cells as male and female mice differ in their responses in this model. Together, these studies clarify the origin of extracellular DNA circulating in the blood in SLE and suggest steps in this process that can be interdicted by novel therapy.

ICF core sets: how to specify impairment and function in systemic lupus erythematosus.

The World Health Organization's International Classification of Function (ICF) is a tool to characterize and illuminate better the full of array of problems a patient faces when affected by disease. Specifying these problems is a particular challenge in a disease like systemic lupus erythematosus (SLE) because of the wide variety in organ systems involved, its variable activity and severity, and considerable ethnic and local differences. The authors of this manuscript believe, however, that a broader understanding will prove essential for optimal patient care, and that there is sufficient experience now in defining ICF Core Sets to successfully complete core sets for SLE. Therefore, we will embark on an international project for developing ICF Core Sets for SLE, which we here delineate. This development will include two versions: 1) The Brief ICF Core Set for SLE will be a very focused list of categories essential for SLE clinical trials; and 2) The Comprehensive ICF Core Set will be much broader and useful for guiding multidisciplinary assessment in patients with SLE. Both Core Sets will be developed in a formal decision-making and consensus process of health professionals integrating evidence gathered from preliminary studies. The final definition of the Core Sets will occur at a consensus conference which will integrate: i) a systematic review of the literature regarding the outcome measures used in clinical trials and selected observational studies; ii) focus groups or semi-structured interviews with SLE patients; iii) a Delphi exercise with world wide involvement of experts; and iv) the evidence from empirical studies. The development of these SLE ICF Core Sets is designed to be an inclusive, open, worldwide process. We therefore invite both SLE clinical experts and SLE patients to participate actively.

The release of microparticles by apoptotic cells and their effects on macrophages.

Microparticles are small membrane vesicles released from the cell membrane by exogenous budding. To elucidate the interactions of microparticles with macrophages, the effect of microparticles released from Jurkat T cells on RAW 264.7 cells was determined. Microparticles were isolated by differential centrifugation, using FACS analysis with annexin V and cell surface markers for identification. Various inducers of apoptosis increased the release of microparticles from Jurkat cells up to 5-fold. The released microparticles were then cultured with RAW 264.7 cells. As shown by confocal microscopy and FACS analysis, RAW 264.7 macrophages cleared microparticles by phagocytosis. In addition, microparticles induced apoptosis in RAW 264.7 cells in a dose-dependent manner with up to a 5-fold increase of annexin V positive cells and 9-fold increase in caspase 3 activity. Cell proliferation as determined by the MTT test was also reduced. Furthermore, microparticles stimulated the release of microparticles from macrophages. These effects were specific for macrophages, since no apoptosis was observed in NIH 3T3 and L929 cells. These findings indicate that microparticles can induce macrophages to undergo apoptosis, in turn resulting in a further increase of microparticles. The release of microparticles from apoptotic cells may therefore represent a novel amplification loop of cell death.

Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro.

DNA is a nuclear macromolecule that circulates in the blood where its levels can reflect the activity of inflammatory and malignant diseases. While dead and dying cells have usually been considered the source of blood DNA, the mechanisms for its release during apoptosis and necrosis are not well defined. To elucidate DNA release, an in vitro model system was used, assessing DNA in the media of living, apoptotic or necrotic Jurkat and U937 cells. Apoptosis was induced by etoposide, camptothecin or staurosporine, while necrosis was induced by heating at 56 degrees C. DNA release was measured by fluorometry with the dye PicoGreen while the extent of death was measured by fluorescence-activated cell sorter analysis with propidium iodide and annexin. Apoptotic Jurkat cells released significantly more DNA in the media than untreated cells while necrotic cells did not show significant DNA release. U937 cells showed similar findings. Pretreatment of Jurkat cells with z-VAD-fmk, a caspase inhibitor, reduced both apoptosis and DNA release. By gel electrophoresis, extracellular DNA from apoptotic cells showed laddering with low molecular weight fragments. These studies suggest that extracellular release of DNA is a consequence of apoptosis and may account for some of the DNA in the blood.

The use of fluorometric assays to assess the immune response to DNA in murine systemic lupus erythematosus.

Antibodies to DNA (anti-DNA) play an important role in the pathogenesis of systemic lupus erythematosus (SLE). In blood, these antibodies may exist in a free, unbound state or as part of complexes with DNA. Furthermore, circulating DNA may be either complexed or free. Because of the central role of these immunoreactants (anti-DNA and DNA) in the disease, monitoring of their levels could provide valuable information for both clinical and investigative purposes. In these studies, we have explored the use of a DNA-binding dye, PicoGreen, for the detection of circulating DNA, either total or immune complex bound. In addition, we have used this dye for Farr-type antibody assays. Using autoimmune MRL/lpr mice as a model, we have shown that, while the levels of free DNA in the plasma of these mice were comparable with those of normal BALB/c mice, the amounts in complexes precipitable by ammonium sulfate were significantly greater. Furthermore, we showed that Farr assays using PicoGreen reliably detect levels of free anti-DNA, with values correlated with anti-DNA levels by an enzyme-linked immunosorbent assay. Together, our results suggest that a fluorometric dye can accurately monitor DNA and anti-DNA antibody levels in SLE and may provide important information on immunopathogenesis.

Induction of cyclooxygenase-2 by mechanical stress through a nitric oxide-regulated pathway.

OBJECTIVE: Biomechanical signals play important roles in regulating the homeostasis of articular cartilage, but under abnormal conditions may be a critical factor in the onset and progression of arthritis. Prostaglandin E(2) (PGE(2)) and nitric oxide (NO), derived from the enzymes cyclo-oxygenase 2 (COX2) and NO synthase 2 (NOS2), are inflammatory mediators that modulate numerous physiological and pathophysiological processes and are potentially important pharmacological targets in osteoarthritis. The goal of this study was to determine the effect of mechanical compression on PGE(2) production in the presence of selective NOS2 and COX2 inhibitors. METHODS: Articular cartilage explants harvested from 2-3-year-old pigs were subjected to intermittent compression at 0.5Hz over a range of stress magnitudes. PGE(2) and NO production into the media were determined in the presence and absence of the NOS2 inhibitor 1400W or the COX2 inhibitor NS398. COX2 protein levels were determined by immunoblot analysis. RESULTS: Mechanical compression significantly increased NO and PGE(2) synthesis in a manner that was dependent on the magnitude of stress. The selective COX2 inhibitor blocked compression-induced NO and PGE(2) production. Compression in the presence of 1400W further increased COX2 expression resulting in a 10-fold increase in PGE(2) production compared to uncompressed explants with 1400W and a 40-fold increase in PGE(2) compared to uncompressed explants without 1400W. CONCLUSION: Mechanical compression of articular cartilage increased COX2 and PGE(2) production through a NO-dependent pathway, and therefore pharmacological agents that target the NOS2 pathway in cartilage may have a significant influence on prostanoid production in the joint.