Publications by authors named "Marc Monestier"

47 Publications

Lymphocyte Activation Gene-3 (LAG-3) negatively regulates environmentally-induced autoimmunity.

PLoS One 2014 14;9(8):e104484. Epub 2014 Aug 14.

Temple Autoimmunity Center and Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.

Environmental factors including drugs, mineral oils and heavy metals such as lead, gold and mercury are triggers of autoimmune diseases in animal models or even in occupationally exposed humans. After exposure to subtoxic levels of mercury (Hg), genetically susceptible strains of mice develop an autoimmune disease characterized by the production of highly specific anti-nucleolar autoantibodies, hyperglobulinemia and nephritis. However, mice can be tolerized to the disease by a single low dose administration of Hg. Lymphocyte Activation Gene-3 (LAG-3) is a CD4-related, MHC-class II binding molecule expressed on activated T cells and NK cells which maintains lymphocyte homeostatic balance via various inhibitory mechanisms. In our model, administration of anti-LAG-3 monoclonal antibody broke tolerance to Hg resulting in autoantibody production and an increase in serum IgE level. In addition, LAG-3-deficient B6.SJL mice not only had increased susceptibility to Hg-induced autoimmunity but were also unresponsive to tolerance induction. Conversely, adoptive transfer of wild-type CD4(+) T cells was able to partially rescue LAG-3-deficient mice from the autoimmune disease. Further, in LAG-3-deficient mice, mercury elicited higher amounts of IL-6, IL-4 and IFN-γ, cytokines known to play a critical role in mercury-induced autoimmunity. Therefore, we conclude that LAG-3 exerts an important regulatory effect on autoimmunity elicited by a common environmental pollutant.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104484PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4133201PMC
April 2015

Efficient clearance of early apoptotic cells by human macrophages requires M2c polarization and MerTK induction.

J Immunol 2012 Oct 31;189(7):3508-20. Epub 2012 Aug 31.

Section of Rheumatology, Department of Medicine, Temple University, Philadelphia, PA 19140, USA.

Mer tyrosine kinase (MerTK) is a major macrophage apoptotic cell (AC) receptor. Its functional impairment promotes autoimmunity and atherosclerosis, whereas overexpression correlates with poor prognosis in cancer. However, little is known about mechanisms regulating MerTK expression in humans. We found that MerTK expression is heterogenous among macrophage subsets, being mostly restricted to anti-inflammatory M2c (CD14(+)CD16(+)CD163(+)CD204(+)CD206(+)CD209(-)) cells, differentiated by M-CSF or glucocorticoids. Small numbers of MerTK(+) "M2c-like" cells are also detectable among circulating CD14(bright)CD16(+) monocytes. MerTK expression levels adapt to changing immunologic environment, being suppressed in M1 and M2a macrophages and in dendritic cells. Remarkably, although glucocorticoid-induced differentiation is IL-10 independent, M-CSF-driven M2c polarization and related MerTK upregulation require IL-10. However, neither IL-10 alone nor TGF-β are sufficient to fully differentiate M2c (CD16(+)CD163(+)MerTK(+)) macrophages. M-CSF and IL-10, both released by T lymphocytes, may thus be required together to promote regulatory T cell-mediated induction of anti-inflammatory monocytes-macrophages. MerTK enables M2c macrophages to clear early ACs more efficiently than other macrophage subsets, and it mediates AC clearance by CD14(bright)CD16(+) monocytes. Moreover, M2c cells release Gas6, which in turn amplifies IL-10 secretion via MerTK. IL-10-dependent induction of the Gas6/MerTK pathway may, therefore, constitute a positive loop for M2c macrophage homeostasis and a critical checkpoint for maintenance of anti-inflammatory conditions. Our findings give new insight into human macrophage polarization and favor a central role for MerTK in regulation of macrophage functions. Eliciting M2c polarization can have therapeutic utility for diseases such as lupus, in which a defective AC clearance contributes to initiate and perpetuate the pathological process.
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http://dx.doi.org/10.4049/jimmunol.1200662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465703PMC
October 2012

Mechanisms of environmental influence on human autoimmunity: a National Institute of Environmental Health Sciences expert panel workshop.

J Autoimmun 2012 Dec 28;39(4):272-84. Epub 2012 Jun 28.

Division of Rheumatology, Allergy, and Clinical Immunology, University of California, Davis, USA.

The mechanisms leading to autoimmune diseases remain largely unknown despite numerous lines of experimental inquiry and epidemiological evidence. The growing number of genome-wide association studies and the largely incomplete concordance for autoimmune diseases in monozygotic twins support the role of the environment (including infectious agents and chemicals) in the breakdown of tolerance leading to autoimmunity via numerous mechanisms. The present article reviews the major theories on the mechanisms of the environmental influence on autoimmunity by addressing the different degrees of confidence that characterize our knowledge. The theories discussed herein include (i) the role of innate immunity mediated by toll-like receptors in triggering the autoimmune adaptive response characterizing the observed pathology; (ii) changes in spleen marginal zone B cells in autoantibody production with particular focus on the B10 subpopulation; (iii) Th17 cell differentiation and T regulatory cells in the aryl hydrocarbon receptor model; (iv) self antigen changes induced by chemical and infectious agents which could break tolerance by post-translational modifications and molecular mimicry; and finally (v) epigenetic changes, particularly DNA methylation, that are induced by environmental stimuli and may contribute to autoimmunity initiation. We are convinced that these working hypotheses, in most cases supported by solid evidence, should be viewed in parallel with animal models and epidemiological observations to provide a comprehensive picture of the environmental causes of autoimmune diseases.
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http://dx.doi.org/10.1016/j.jaut.2012.05.007DOI Listing
December 2012

Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury.

J Clin Invest 2012 Jul 11;122(7):2661-71. Epub 2012 Jun 11.

Cardiovascular Research Institute, UCSF, San Francisco, CA, USA

There is emerging evidence that platelets are major contributors to inflammatory processes through intimate associations with innate immune cells. Here, we report that activated platelets induce the formation of neutrophil extracellular traps (NETs) in transfusion-related acute lung injury (TRALI), which is the leading cause of death after transfusion therapy. NETs are composed of decondensed chromatin decorated with granular proteins that function to trap extracellular pathogens; their formation requires the activation of neutrophils and release of their DNA in a process that may or may not result in neutrophil death. In a mouse model of TRALI that is neutrophil and platelet dependent, NETs appeared in the lung microvasculature and NET components increased in the plasma. We detected NETs in the lungs and plasma of human TRALI and in the plasma of patients with acute lung injury. In the experimental TRALI model, targeting platelet activation with either aspirin or a glycoprotein IIb/IIIa inhibitor decreased NET formation and lung injury. We then directly targeted NET components with a histone blocking antibody and DNase1, both of which protected mice from TRALI. These data suggest that NETs contribute to lung endothelial injury and that targeting NET formation may be a promising new direction for the treatment of acute lung injury.
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http://dx.doi.org/10.1172/JCI61303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3386815PMC
July 2012

Histones from dying renal cells aggravate kidney injury via TLR2 and TLR4.

J Am Soc Nephrol 2012 Aug 7;23(8):1375-88. Epub 2012 Jun 7.

Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Pettenkoferstr. 8a, D-80336 Munich, Germany.

In AKI, dying renal cells release intracellular molecules that stimulate immune cells to secrete proinflammatory cytokines, which trigger leukocyte recruitment and renal inflammation. Whether the release of histones, specifically, from dying cells contributes to the inflammation of AKI is unknown. In this study, we found that dying tubular epithelial cells released histones into the extracellular space, which directly interacted with Toll-like receptor (TLR)-2 (TLR2) and TLR4 to induce MyD88, NF-κB, and mitogen activated protein kinase signaling. Extracellular histones also had directly toxic effects on renal endothelial cells and tubular epithelial cells in vitro. In addition, direct injection of histones into the renal arteries of mice demonstrated that histones induce leukocyte recruitment, microvascular vascular leakage, renal inflammation, and structural features of AKI in a TLR2/TLR4-dependent manner. Antihistone IgG, which neutralizes the immunostimulatory effects of histones, suppressed intrarenal inflammation, neutrophil infiltration, and tubular cell necrosis and improved excretory renal function. In summary, the release of histones from dying cells aggravates AKI via both its direct toxicity to renal cells and its proinflammatory effects. Because the induction of proinflammatory cytokines in dendritic cells requires TLR2 and TLR4, these results support the concept that renal damage triggers an innate immune response, which contributes to the pathogenesis of AKI.
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http://dx.doi.org/10.1681/ASN.2011111077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402284PMC
August 2012

Neutrophil cerebrovascular transmigration triggers rapid neurotoxicity through release of proteases associated with decondensed DNA.

J Immunol 2012 Jul 1;189(1):381-92. Epub 2012 Jun 1.

Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.

Cerebrovascular inflammation contributes to diverse CNS disorders through mechanisms that are incompletely understood. The recruitment of neutrophils to the brain can contribute to neurotoxicity, particularly during acute brain injuries, such as cerebral ischemia, trauma, and seizures. However, the regulatory and effector mechanisms that underlie neutrophil-mediated neurotoxicity are poorly understood. In this study, we show that mouse neutrophils are not inherently toxic to neurons but that transendothelial migration across IL-1-stimulated brain endothelium triggers neutrophils to acquire a neurotoxic phenotype that causes the rapid death of cultured neurons. Neurotoxicity was induced by the addition of transmigrated neutrophils or conditioned medium, taken from transmigrated neutrophils, to neurons and was partially mediated by excitotoxic mechanisms and soluble proteins. Transmigrated neutrophils also released decondensed DNA associated with proteases, which are known as neutrophil extracellular traps. The blockade of histone-DNA complexes attenuated transmigrated neutrophil-induced neuronal death, whereas the inhibition of key neutrophil proteases in the presence of transmigrated neutrophils rescued neuronal viability. We also show that neutrophil recruitment in the brain is IL-1 dependent, and release of proteases and decondensed DNA from recruited neutrophils in the brain occurs in several in vivo experimental models of neuroinflammation. These data reveal new regulatory and effector mechanisms of neutrophil-mediated neurotoxicity (i.e., the release of proteases and decondensed DNA triggered by phenotypic transformation during cerebrovascular transmigration). Such mechanisms have important implications for neuroinflammatory disorders, notably in the development of antileukocyte therapies.
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http://dx.doi.org/10.4049/jimmunol.1200409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381844PMC
July 2012

Extracellular chromatin is an important mediator of ischemic stroke in mice.

Arterioscler Thromb Vasc Biol 2012 Aug 24;32(8):1884-91. Epub 2012 May 24.

Immune Disease Institute, Harvard Medical School, 3 Blackfan Circle, Boston, MA 02115, USA.

Objective: Recently, a growing number of studies have revealed a prothrombotic and cytotoxic role for extracellular chromatin. Cerebral ischemia/reperfusion injury is characterized by a significant amount of cell death and neutrophil activation, both of which may result in the release of chromatin. The goal of this study was to assess the effect of extracellular chromatin in ischemic stroke using a mouse model of transient middle cerebral artery occlusion.

Methods And Results: Similar to reports in stroke patients, we observed increased levels of circulating nucleosomes and DNA after ischemic stroke in mice. In addition, we observed that general hypoxia also augmented extracellular chromatin. We hypothesized that targeting extracellular chromatin components would be protective in ischemic stroke. Indeed, treatment with recombinant human DNase 1 significantly improved stroke outcome. Neutralization of histones using an antihistone antibody was also protective as evidenced by smaller infarct volumes, whereas increasing levels of extracellular histones via histone infusion exacerbated stroke outcome by increasing infarct size and worsening functional outcome.

Conclusions: Our results indicate that extracellular chromatin is generated and is detrimental during cerebral ischemia/reperfusion in mice. Targeting DNA and histones may be a new therapeutic strategy to limit injury resulting from ischemic stroke.
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http://dx.doi.org/10.1161/ATVBAHA.112.250993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3494463PMC
August 2012

Detection of extracellular genomic DNA scaffold in human thrombus: implications for the use of deoxyribonuclease enzymes in thrombolysis.

J Vasc Interv Radiol 2012 May;23(5):712-8

Division of Vascular Imaging and Intervention, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.

Purpose: Mechanisms underlying transition of a thrombus susceptible to tissue plasminogen activator (TPA) fibrinolysis to one that is resistant is unclear. Demonstration of a new possible thrombus scaffold may open new avenues of research in thrombolysis and may provide mechanistic insight into thrombus remodeling.

Materials And Methods: Ten human thrombus samples were collected during cases of thrombectomy and open surgical repair of abdominal aortic aneurysms (five samples < 3 d old and five samples > 1 y old). Additionally, an acute murine hindlimb ischemia model was created to evaluate thrombus samples in mice. Human sections were immunostained for the H2A/H2B/DNA complex, myeloperoxidase, fibrinogen, and von Willebrand factor. Mouse sections were immunostained with the H2A antibody. All samples were further evaluated after hematoxylin and eosin and Masson trichrome staining.

Results: An extensive network of extracellular histone/DNA complex was demonstrated in the matrix of human ex vivo thrombus. This network is present throughout the highly cellular acute thrombus. However, in chronic thrombi, detection of the histone/DNA network was predominantly in regions of low collagen content and high cell density, which were mostly near the lumen. These regions of high cell density contained neutrophils and monocytes. Similarly, sections from the acute murine hindlimb ischemia model also exhibited extensive immunoreactivity to the histone antibody in the extracellular space within murine thrombi.

Conclusions: Extensive detection of genomic DNA associated with histones in the extracellular matrix of human and mouse thrombi suggest the presence of a new thrombus-associated scaffold.
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http://dx.doi.org/10.1016/j.jvir.2012.01.072DOI Listing
May 2012

Plasmodium falciparum histones induce endothelial proinflammatory response and barrier dysfunction.

Am J Pathol 2012 Mar 17;180(3):1028-1039. Epub 2012 Jan 17.

Department of Microbiology Immunology & Infectious Diseases, University of Calgary, Alberta, Canada. Electronic address:

Plasmodium falciparum is a protozoan parasite of human erythrocytes that causes the most severe form of malaria. Severe P. falciparum infection is associated with endothelial activation and permeability, which are important determinants of the outcome of the infection. How endothelial cells become activated is not fully understood, particularly with regard to the effects of parasite subcomponents. We demonstrated that P. falciparum histones extracted from merozoites (HeH) directly stimulated the production of IL-8 and other inflammatory mediators by primary human dermal microvascular endothelial cells through a signaling pathway that involves Src family kinases and p38 MAPK. The stimulatory effect of HeH and recombinant P. falciparum H3 (PfH3) was abrogated by histone-specific antibodies. The release of nuclear contents on rupture of infected erythrocytes was captured by live cell imaging and confirmed by detecting nucleosomes in the supernatants of parasite cultures. HeH and recombinant parasite histones also induced endothelial permeability through a charge-dependent mechanism that resulted in disruption of junctional protein expression and cell death. Recombinant human activated protein C cleaved HeH and PfH3 and abrogated their proinflammatory effects. Circulating nucleosomes of both human and parasite origin were detected in the plasma of patients with falciparum malaria and correlated positively with disease severity. These results support a pathogenic role for both host- and pathogen-derived histones in P. falciparum-caused malaria.
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http://dx.doi.org/10.1016/j.ajpath.2011.11.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448071PMC
March 2012

Caspase-activated DNase is required for maintenance of tolerance to lupus nuclear autoantigens.

Arthritis Rheum 2012 Apr 29;64(4):1247-56. Epub 2011 Nov 29.

Temple University, Philadelphia, Pennsylvania 19140, USA.

Objective: Caspase-activated DNase (CAD) is an endonuclease that is activated by active caspase 3 during apoptosis and is responsible for degradation of chromatin into nucleosomal units. These nucleosomal units are then included in apoptotic bodies. The presence of apoptotic bodies is considered important for the generation of autoantigen in autoimmune diseases, such as systemic lupus erythematosus (SLE), that are characterized by the presence of antinuclear antibodies. The present study was carried out to determine the role of CAD in SLE and to investigate the ability of lupus autoantibodies to bind to CAD-deficient or CAD-sufficient apoptotic cells.

Methods: The Sle1, Sle123, and 3H9 mouse models of SLE, in which autoimmunity is genetically predetermined, were used. To determine the role of chromatin fragmentation in SLE, CAD deficiency was introduced in these mouse models.

Results: Deficiency of CAD resulted in increased anti-double-stranded DNA antibody titers in lupus-prone mice. Surprisingly, the absence of CAD exacerbated only genetically predetermined autoimmune responses. To further determine whether nuclear modifications are needed in order to maintain tolerance to nuclear autoantigens, we used the 3H9 mouse, an anti-DNA heavy chain knockin; in this model, the autoreactive B cells are tolerized by anergy. In accordance with findings in the CAD-mutant Sle1 and Sle123 mice, CAD-deficient 3H9 mice spontaneously generated anti-DNA antibodies. Finally, we showed that autoantibodies with specificities toward histone-DNA complexes bind more to CAD-deficient apoptotic cells than to CAD-sufficient apoptotic cells.

Conclusion: We propose that in mice that are genetically predisposed to lupus development, nuclear apoptotic modifications are needed to maintain tolerance. In the absence of these modifications, apoptotic chromatin is abnormally exposed, facilitating the autoimmune response.
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http://dx.doi.org/10.1002/art.33448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292632PMC
April 2012

Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury.

J Immunol 2011 Sep 22;187(5):2626-31. Epub 2011 Jul 22.

Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.

We previously reported that extracellular histones are major mediators of death in sepsis. Infusion of extracellular histones leads to increased cytokine levels. Histones activate TLR2 and TLR4 in a process that is enhanced by binding to DNA. Activation of TLR4 is responsible for the histone-dependent increase in cytokine levels. To study the impact of histone release on pathology we used two models: a Con A-triggered activation of T cells to mimic sterile inflammation, and acetaminophen to model drug-induced tissue toxicity. Histones were released in both models and anti-histone Abs were protective. TLR2- or TLR4-null mice were also protected. These studies imply that histone release contributes to death in inflammatory injury and in chemical-induced cellular injury, both of which are mediated in part through the TLRs.
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http://dx.doi.org/10.4049/jimmunol.1003930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159755PMC
September 2011

An epichromatin epitope: persistence in the cell cycle and conservation in evolution.

Nucleus 2011 Jan-Feb;2(1):47-60

Department of Pharmaceutical Sciences, College of Pharmacy, University of New England, Portland, ME USA. ted proteins (ARPs), a

Interphase nuclear architecture is disrupted and rapidly reformed with each cell division cycle. Successive cell generations exhibit a "memory" of this nuclear architecture, as well as for gene expression. Furthermore, many features of nuclear and mitotic chromosome structure are recognizably species and tissue specific. We wish to know what properties of the underlying chromatin structure may determine these conserved features of nuclear architecture. Employing a particular mouse autoimmune anti-nucleosome monoclonal antibody (PL2-6), combined with deconvolution immunofluorescence microscopy, we present evidence for a unique epitope (involving a ternary complex of histones H2A and H2B and DNA) which is localized only at the exterior chromatin surface of interphase nuclei and mitotic chromosomes in mammalian, invertebrate and plant systems. As only the surface chromatin region is identified with antibody PL2-6, we have assigned it the name "epichromatin". We describe an "epichromatin hypothesis", suggesting that epichromatin may have a unique evolutionary conserved conformation which facilitates interaction with the reforming post-mitotic nuclear envelope and a rapid return of interphase nuclear architecture.
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http://dx.doi.org/10.4161/nucl.2.1.13271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3104809PMC
January 2012

Microparticles as antigenic targets of antibodies to DNA and nucleosomes in systemic lupus erythematosus.

J Autoimmun 2011 May 3;36(3-4):173-80. Epub 2011 Mar 3.

Duke University Medical Center, Department of Medicine, Durham, NC 27705, USA.

Systemic lupus erythematosus is a prototypic autoimmune disease characterized by antibodies to DNA and other nuclear molecules. While these antibodies can form immune complexes, the mechanisms generating the bound nuclear antigens are not known. These studies investigated whether microparticles can form complexes with anti-DNA and other anti-nucleosomal antibodies. Microparticles are small membrane-bound vesicles released from dead and dying cells; these particles contain a variety of cellular components, including DNA. To assess antigenicity, microparticles generated in vitro from apoptotic cell lines were tested using murine monoclonal anti-DNA and anti-nucleosomal antibodies as well as plasma from lupus patients. Antibody binding was assessed by flow cytometry. As these studies showed, some but not all of the monoclonal antibodies bound to microparticles prepared from apoptotic HL-60, THP-1 and Jurkat cells. For HL-60 cells, both staurosporine and UV radiation led to the production of antigenically active particles. For the anti-DNA antibody with high particle binding, prior treatment of DNase reduced activity. With plasma from patients with SLE, antibody binding to microparticles was present although a clear relationship with anti-DNA antibody levels was not observed. To determine whether lupus plasma contains immune complexes with particle properties, particle preparations were tested for bound IgG by flow cytometry. These studies indicated that lupus plasma contains particles with IgG binding, with numbers correlated with anti-DNA levels. Together, these findings indicate that microparticles display DNA and nucleosomal molecules in an antigenic form and could represent a source of immune complexes in SLE.
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http://dx.doi.org/10.1016/j.jaut.2011.02.001DOI Listing
May 2011

B cell receptor editing in tolerance and autoimmunity.

Ann N Y Acad Sci 2011 Jan 5;1217:96-121. Epub 2011 Jan 5.

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

Receptor editing is the process of ongoing antibody gene rearrangement in a lymphocyte that already has a functional antigen receptor. The expression of a functional antigen receptor will normally terminate further rearrangement (allelic exclusion). However, lymphocytes with autoreactive receptors have a chance at escaping negative regulation by "editing" the specificities of their receptors with additional antibody gene rearrangements. As such, editing complicates the Clonal Selection Hypothesis because edited cells are not simply endowed for life with a single, invariant antigen receptor. Furthermore, if the initial immunoglobulin gene is not inactivated during the editing process, allelic exclusion is violated and the B cell can exhibit two specificities. Here, we describe the discovery of editing, the pathways of receptor editing at the heavy (H) and light (L) chain loci, and current evidence regarding how and where editing happens and what effects it has on the antibody repertoire.
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http://dx.doi.org/10.1111/j.1749-6632.2010.05877.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077556PMC
January 2011

Statins enhance formation of phagocyte extracellular traps.

Cell Host Microbe 2010 Nov;8(5):445-54

Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA.

Statins are inhibitors of 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis. Recent clinico-epidemiologic studies correlate patients receiving statin therapy with having reduced mortality associated with severe bacterial infection. Investigating the effect of statins on the innate immune capacity of phagocytic cells against the human pathogen Staphylococcus aureus, we uncovered a beneficial effect of statins on bacterial clearance by phagocytes, although, paradoxically, both phagocytosis and oxidative burst were inhibited. Probing instead for an extracellular mechanism of killing, we found that statins boosted the production of antibacterial DNA-based extracellular traps (ETs) by human and murine neutrophils and also monocytes/macrophages. The effect of statins to induce phagocyte ETs was linked to sterol pathway inhibition. We conclude that a drug therapy taken chronically by millions alters the functional behavior of phagocytic cells, which could have ramifications for susceptibility and response to bacterial infections in these patients.
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http://dx.doi.org/10.1016/j.chom.2010.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3008410PMC
November 2010

Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps.

J Innate Immun 2010 10;2(6):576-86. Epub 2010 Sep 10.

Department of Pediatrics, University of California San Diego, La Jolla, CA, USA.

Neutrophils are key effectors of the host innate immune response against bacterial infection. Staphylococcus aureus is a preeminent human pathogen, with an ability to produce systemic infections even in previously healthy individuals, thereby reflecting a resistance to effective neutrophil clearance. The recent discovery of neutrophil extracellular traps (NETs) has opened a novel dimension in our understanding of how these specialized leukocytes kill pathogens. NETs consist of a nuclear DNA backbone associated with antimicrobial peptides, histones and proteases that provide a matrix to entrap and kill various microbes. Here, we used targeted mutagenesis to examine a potential role of S. aureus nuclease in NET degradation and virulence in a murine respiratory tract infection model. In vitro assays using fluorescence microscopy showed the isogenic nuclease-deficient (nuc-deficient) mutant to be significantly impaired in its ability to degrade NETs compared with the wild-type parent strain USA 300 LAC. Consequently, the nuc-deficient mutant strain was significantly more susceptible to extracellular killing by activated neutrophils. Moreover, S. aureus nuclease production was associated with delayed bacterial clearance in the lung and increased mortality after intranasal infection. In conclusion, this study shows that S. aureus nuclease promotes resistance against NET-mediated antimicrobial activity of neutrophils and contributes to disease pathogenesis in vivo.
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http://dx.doi.org/10.1159/000319909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2982853PMC
May 2011

Extracellular DNA traps promote thrombosis.

Proc Natl Acad Sci U S A 2010 Sep 23;107(36):15880-5. Epub 2010 Aug 23.

Immune Disease Institute, Boston, MA 02115, USA.

Neutrophil extracellular traps (NETs) are part of the innate immune response to infections. NETs are a meshwork of DNA fibers comprising histones and antimicrobial proteins. Microbes are immobilized in NETs and encounter a locally high and lethal concentration of effector proteins. Recent studies show that NETs are formed inside the vasculature in infections and noninfectious diseases. Here we report that NETs provide a heretofore unrecognized scaffold and stimulus for thrombus formation. NETs perfused with blood caused platelet adhesion, activation, and aggregation. DNase or the anticoagulant heparin dismantled the NET scaffold and prevented thrombus formation. Stimulation of platelets with purified histones was sufficient for aggregation. NETs recruited red blood cells, promoted fibrin deposition, and induced a red thrombus, such as that found in veins. Markers of extracellular DNA traps were detected in a thrombus and plasma of baboons subjected to deep vein thrombosis, an example of inflammation-enhanced thrombosis. Our observations indicate that NETs are a previously unrecognized link between inflammation and thrombosis and may further explain the epidemiological association of infection with thrombosis.
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http://dx.doi.org/10.1073/pnas.1005743107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2936604PMC
September 2010

Reversal of established lupus nephritis and prolonged survival of New Zealand black x New Zealand white mice treated with the topoisomerase I inhibitor irinotecan.

J Immunol 2010 Feb 18;184(4):2175-82. Epub 2010 Jan 18.

Department of Clinical Research, University Hospital Bern, Bern, Switzerland.

Systemic lupus erythematosus is a chronic autoimmune disorder that predominantly affects women of childbearing age. Lupus-associated glomerulonephritis is a major cause of mortality in these patients. Current treatment protocols for systemic lupus erythematosus include cyclophosphamide, prednisolone, azathioprine, and mycophenolate mofetil. However, in mice none of these agents alone or in combination were shown to reverse established proteinuria. Using New Zealand Black x New Zealand White F1 mice, we report that administration of the topoisomerase I inhibitor irinotecan from week 13 completely prevented the onset of proteinuria and prolonged survival up to at least 90 wk without detectable side effects. Furthermore, application of irinotecan to mice with established lupus nephritis, as indicated by grade 3+ (> or =300 mg/dl) and grade 4+ (> or =2000 mg/dl) proteinuria and, according to a median age of 35 wk, resulted in remission rates of 75% and 55%, respectively. Survival was significantly prolonged with 73 wk (grade 3+ and 4+ combined) versus 40 wk for control animals. Although total IgG and anti-dsDNA Abs in the serum and mesangial IgG deposits in the kidneys were not reduced in irinotecan-treated mice, subendothelial immune deposits were considerably diminished, suggesting a prevention of glomerular basement membrane disruption. This effect was accompanied by increased rates of ssDNA breaks and inhibition of renal cell apoptosis being different to what is known about irinotecan in anticancer therapy. In conclusion, our data provide evidence that irinotecan might represent an entirely new strategy for the treatment of systemic lupus erythematosus.
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http://dx.doi.org/10.4049/jimmunol.0903153DOI Listing
February 2010

Extracellular histones are major mediators of death in sepsis.

Nat Med 2009 Nov 25;15(11):1318-21. Epub 2009 Oct 25.

Oklahoma Medical Research Foundation, Oklahoma City, OK, USA.

Hyperinflammatory responses can lead to a variety of diseases, including sepsis. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra-alveolar hemorrhage and macro- and microvascular thrombosis. We detected histone in the circulation of baboons challenged with Escherichia coli, and the increase in histone levels was accompanied by the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co-infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality, which was reversed by treatment with antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.
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http://dx.doi.org/10.1038/nm.2053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2783754PMC
November 2009

Apoptosis-associated acetylation on histone H2B is an epitope for lupus autoantibodies.

Mol Immunol 2009 Dec 10;47(2-3):511-6. Epub 2009 Sep 10.

Nephrology Research Laboratory, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands.

Objective: Nucleosomes have been identified as a key autoantigen in systemic lupus erythematosus (SLE). Nucleosomes are present in the circulation due to a disturbed apoptosis and/or an insufficient clearance in SLE. During apoptosis, histones can be modified, thereby making them more immunogenic. Recently, we showed the importance of apoptosis-induced acetylation of histone H4 in the pathogenesis of SLE. The lupus-derived antibody LG11-2 was previously shown to react with the N-terminal tail of histone H2B, which contains amino acid residues that can be modified including phosphorylation of serine 14, known to occur during apoptosis. Here, we evaluate whether apoptosis-induced histone modifications on H2B exist that are targeted by LG11-2 or lupus-derived plasmas.

Methods: Immunofluorescence staining and western immunoblot analysis of control, apoptotic and trichostatin A-treated cells/chromatin were performed with monoclonal antibody LG11-2. Reactivity of LG11-2 and plasmas from lupus mice and SLE patients with acetylated and/or phosphorylated H2B peptides was determined in competition ELISA.

Results: LG11-2 showed enhanced reactivity with apoptotic and hyperacetylated H2B compared to normal H2B. This enhanced reactivity was due to the acetylation of lysine 12 in H2B. This modification was also recognized by autoantibodies from pre-diseased lupus mice, but to a lesser extent by plasmas of diseased lupus mice and lupus patients.

Conclusions: The apoptosis-induced acetylation on H2BK12 is a target for autoantibodies in SLE. Since the anti-H2BK12ac reactivity was mainly found in pre-diseased lupus mice, this epitope seems important in the early phase of the anti-chromatin autoimmune response with subsequent epitope spreading to unmodified H2B.
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http://dx.doi.org/10.1016/j.molimm.2009.08.009DOI Listing
December 2009

A broad screen for targets of immune complexes decorating arthritic joints highlights deposition of nucleosomes in rheumatoid arthritis.

Proc Natl Acad Sci U S A 2009 Sep 31;106(37):15867-72. Epub 2009 Aug 31.

Section on Immunology and Immunogenetics, The Joslin Diabetes Center, Boston, Massachusetts, USA.

Deposits of Ig and complement are abundant in affected joints of patients with rheumatoid arthritis (RA) and in animal models of RA in which antibodies are demonstrably pathogenic. To identify molecular targets of the Igs deposited in arthritic joints, which may activate local inflammation, we used a combination of mass spectrometry (MS) and protein microarrays. Immune complexes were affinity-purified from surgically removed joint tissues of 26 RA and osteoarthritis (OA) patients. Proteins complexed with IgG were identified by proteomic analysis using tandem MS. A striking diversity of components of the extracellular matrix, and some intracellular components, copurified specifically with IgG from RA and OA tissues. A smaller set of autoantigens was observed only in RA eluates. In complementary experiments, IgG fractions purified from joint immune complexes were tested on protein microarrays against a range of candidate autoantigens. These Igs bound a diverse subset of proteins and peptides from synovium and cartilage, different from that bound by normal serum Ig. One type of intracellular protein detected specifically in RA joints (histones H2A/B) was validated by immunohistology and found to be deposited on the cartilage surface of RA but not OA joints. Thus, autoantibodies to many determinants (whether deposited as "neoantigens" or normal constituents of the extracellular matrix) have the potential to contribute to arthritic inflammation.
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http://dx.doi.org/10.1073/pnas.0908032106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2747210PMC
September 2009

How can a chemical element elicit complex immunopathology? Lessons from mercury-induced autoimmunity.

Trends Immunol 2009 Oct 24;30(10):502-9. Epub 2009 Aug 24.

Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania, 19140, United States.

Although most autoimmune diseases develop without a manifest cause, epidemiological studies indicate that external factors play an important role in triggering or aggravating autoimmune processes in genetically predisposed individuals. Nevertheless, most autoimmune disease-promoting environmental agents are unknown because their relationships to immune function are not understood. Thus, the study of animal models of chemically-induced autoimmunity should shed light on the pathways involved and allow us to identify these agents. The rodent model of heavy metal-induced autoimmunity is one of the most intriguing experimental systems available to address such questions. Although the ultimate pathophysiology of this model remains mysterious, recent studies have started to elucidate the mechanisms by which heavy metal exposure leads to immune activation and loss of self-tolerance.
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http://dx.doi.org/10.1016/j.it.2009.07.005DOI Listing
October 2009

Requirement for DNA CpG content in TLR9-dependent dendritic cell activation induced by DNA-containing immune complexes.

J Immunol 2009 Sep 31;183(5):3109-17. Epub 2009 Jul 31.

Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.

Although TLR9 was originally thought to specifically recognize microbial DNA, it is now evident that mammalian DNA can be an effective TLR9 ligand. However, the DNA sequence required for TLR9 activation is controversial, as studies have shown conflicting results depending on the nature of the DNA backbone, the route of DNA uptake, and the cell type being studied. In systemic lupus erythematosus, a major route whereby DNA gains access to intracellular TLR9, and thereby activates dendritic cells (DCs), is through uptake as a DNA-containing immune complex. In this report, we used defined dsDNA fragments with a natural (phosphodiester) backbone and show that unmethylated CpG dinucleotides within dsDNA are required for murine DC TLR9 activation induced by a DNA-containing immune complex. The strongest activation is seen with dsDNA fragments containing optimal CpG motifs (purine-purine-CpG-pyrimidine-pyrimidine) that are common in microbial DNA but rare in mammalian DNA. Importantly, however, activation can also be induced by CpG-rich DNA fragments that lack these optimal CpG motifs and that we show are plentiful in CpG islands within mammalian DNA. No activation is induced by DNA fragments lacking CpG dinucleotides, although this CpG-free DNA can induce DC activation if internalized by liposomal transfection instead of as an immune complex. Overall, the data suggest that the release of CpG-rich DNA from mammalian DNA may contribute to the pathogenesis of autoimmune diseases such as systemic lupus erythematosus and psoriasis in which activation of TLR9 in DCs by self DNA has been implicated in disease pathogenesis.
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http://dx.doi.org/10.4049/jimmunol.0900399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860771PMC
September 2009

Histone H4 is a major component of the antimicrobial action of human sebocytes.

J Invest Dermatol 2009 Oct 18;129(10):2489-96. Epub 2009 Jun 18.

Division of Dermatology, University of California San Diego and VA San Diego Healthcare Center, San Diego, California, USA.

Antimicrobial peptides, such as cathelicidin and beta defensins, directly kill microbes and have been detected in human sebaceous glands and cell lines. Despite the presence of several such peptides, the apparent abundance of these is insufficient for direct killing of most skin pathogens. In this study, we sought to determine which molecules provide the majority of antimicrobial peptide activity in human sebocytes. Acid-soluble protein extracts of SEB-1 sebocytes were separated by reverse-phase high-performance liquid chromatography and were assayed for their capacity to inhibit the growth of Staphylococcus aureus. Antimicrobial activity was isolated in a single major fraction and identified to be histone H4 by mass spectrometry and western blot analysis. The importance of histone H4 in the antimicrobial activity of sebocytes was confirmed by a specific neutralizing antibody and by direct demonstration that recombinant histone H4 had antimicrobial activity against S. aureus and Propionibacterium acnes. In addition, histone H4 enhanced the antimicrobial action of free fatty acids in human sebum. Taken together, these results indicate that the release of histone H4 by holocrine secretion from the sebaceous gland may play an important role in innate immunity.
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http://dx.doi.org/10.1038/jid.2009.106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2765036PMC
October 2009

Role of tissue factor in a mouse model of thrombotic microangiopathy induced by antiphospholipid antibodies.

Blood 2009 Aug 17;114(8):1675-83. Epub 2009 Jun 17.

Department of Pathology, Department of Medicine, Weill Cornell Medical College, New York, NY, USA.

Using different mouse monoclonal and human antiphospholipid (aPL) antibodies, we developed a new animal model of renal injury that shares many features with thrombotic microangiopathy (TMA). We found that more than 1 mechanism/signaling pathway is involved in glomerular injury induced by aPL antibodies in this model. Both complement-dependent and complement-independent pathways were identified that lead to glomerular endothelial cell damage and renal function impairment. We also found that C5a-C5aR interaction is a crucial step for the activation of the coagulation cascade and glomerular injury induced by complement-activating antibodies. In addition, our studies demonstrated complement-independent mechanisms in which reactivity with beta(2) glycoprotein I (beta2GPI) plays an important role in aPL-induced glomerular damage and renal failure. Independently of the mechanism responsible for aPL-induced TMA, mice that express low levels of tissue factor (TF) were protected from glomerular injury. That genetic reduction of TF prevents renal injury induced by different aPL antibodies indicates that TF is a common mediator of glomerular damage and a possible target for selective pharmacologic intervention. Treatment with pravastatin, which down-regulates glomerular TF synthesis, prevents aPL-induced TMA in this mouse model, thus emphasizing that targeting TF might be a good therapeutic intervention in patients with TMA.
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http://dx.doi.org/10.1182/blood-2009-01-199117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2927091PMC
August 2009

Immunology of mercury.

Ann N Y Acad Sci 2008 Nov;1143:240-67

Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140, USA.

The heavy metal mercury is ubiquitously distributed in the environment resulting in permanent low-level exposure in human populations. Mercury can be encountered in three main chemical forms (elemental, inorganic, and organic) which can affect the immune system in different ways. In this review, we describe the effects of these various forms of mercury exposure on immune cells in humans and animals. In genetically susceptible mice or rats, subtoxic doses of mercury induce the production of highly specific autoantibodies as well as a generalized activation of the immune system. We review studies performed in this model and discuss their implications for the role of environmental chemicals in human autoimmunity.
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http://dx.doi.org/10.1196/annals.1443.022DOI Listing
November 2008

Regulatory roles for NKT cell ligands in environmentally induced autoimmunity.

J Immunol 2008 Nov;181(10):6779-88

Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140, USA.

The development of autoimmune diseases is frequently linked to exposure to environmental factors such as chemicals, drugs, or infections. In the experimental model of metal-induced autoimmunity, administration of subtoxic doses of mercury (a common environmental pollutant) to genetically susceptible mice induces an autoimmune syndrome with rapid anti-nucleolar Ab production and immune system activation. Regulatory components of the innate immune system such as NKT cells and TLRs can also modulate the autoimmune process. We examined the interplay among environmental chemicals and NKT cells in the regulation of autoimmunity. Additionally, we studied NKT and TLR ligands in a tolerance model in which preadministration of a low dose of mercury in the steady state renders animals tolerant to metal-induced autoimmunity. We also studied the effect of Sphingomonas capsulata, a bacterial strain that carries both NKT cell and TLR ligands, on metal-induced autoimmunity. Overall, NKT cell activation by synthetic ligands enhanced the manifestations of metal-induced autoimmunity. Exposure to S. capsulata exacerbated autoimmunity elicited by mercury. Although the synthetic NKT cell ligands that we used are reportedly similar in their ability to activate NKT cells, they displayed pronounced differences when coinjected with environmental agents or TLR ligands. Individual NKT ligands differed in their ability to prevent or break tolerance induced by low-dose mercury treatment. Likewise, different NKT ligands either dramatically potentiated or inhibited the ability of TLR9 agonistic oligonucleotides to disrupt tolerance to mercury. Our data suggest that these differences could be mediated by the modification of cytokine profiles and regulatory T cell numbers.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647253PMC
http://dx.doi.org/10.4049/jimmunol.181.10.6779DOI Listing
November 2008

The human granulocyte nucleus: Unusual nuclear envelope and heterochromatin composition.

Eur J Cell Biol 2008 May 8;87(5):279-90. Epub 2008 Apr 8.

Department of Biology, Bowdoin College, Brunswick, ME 04011, USA.

The human blood granulocyte (neutrophil) is adapted to find and destroy infectious agents. The nucleus of the human neutrophil has a segmented appearance, consisting of a linear or branched array of three or four lobes. Adequate levels of lamin B receptor (LBR) are necessary for differentiation of the lobulated nucleus. The levels of other components of the nuclear envelope may also be important for nuclear shape determination. In the present study, immunostaining and immunoblotting procedures explored the levels of various components of the nuclear envelope and heterochromatin, comparing freshly isolated human neutrophils with granulocytic forms of HL-60 cells, a tissue culture model system. In comparison to granulocytic HL-60 cells, blood neutrophil nuclear envelopes contain low-to-negligible amounts of LBR, lamins A/C, B1 and B2, LAP2beta and emerin. Surprisingly, a "mitotic" chromosome marker, H3(S10)phos, is elevated in neutrophil nuclei, compared to granulocytic HL-60 cells. Furthermore, neutrophil nuclei appear to be more fragile to methanol fixation, than observed with granulocytic HL-60 cells. Thus, the human neutrophil nucleus appears to be highly specialized, possessing a paucity of nuclear envelope-stabilizing proteins. In consequence, the neutrophil nucleus appears to be very malleable, supporting rapid migration through tight tissue spaces.
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http://dx.doi.org/10.1016/j.ejcb.2008.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2438038PMC
May 2008

Accelerated atherosclerosis in ApoE deficient lupus mouse models.

Clin Immunol 2008 May 5;127(2):168-75. Epub 2008 Mar 5.

Divisions of Rheumatology, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

The accelerated development of atherosclerosis with increased risk of cardiovascular disease in systemic lupus erythematosus (SLE) patients is not well understood. An appropriate mouse model would greatly help to understand the mechanisms of this association. We have therefore combined the ApoE(-/-) model of atherosclerosis with three different murine models of SLE. We found that induction of cGVH in B6.ApoE(-/-) mice, breeding a Fas null gene onto the B6.ApoE(-/-) mice, and breeding the ApoE(-/-) defect onto MRL/lpr mice all caused a modest increase of atherosclerosis at 24 weeks of age compared to B6.ApoE(-/-) controls. B cells in B6.ApoE(-/-) mice had certain phenotypic differences compared to congenic C57BL/6 mice, as indicated by high expression of MHC II, Fas, CD86, and by increased number of cells bearing marginal zone phenotype. Furthermore, B6ApoE(-/-) mice had significant titers of anti-oxLDL and anti-cardiolipin autoantibodies compared to their B6 counterparts. Our studies also indicate that, following induction of cGVH, marginal zone B cells in B6.ApoE(-/-) are depleted, and there is considerable increase in anti-oxLDL and anti-cardiolipin abs along with secretion of lupus-specific autoantibodies, such as anti-dsDNA and anti-chromatin abs. Histological sections showed that cGVH and/or Fas deficiency could exacerbate atherosclerosis. The production of anti-oxLDL and anti-cardiolipin in ApoE(-/-) mice was also increased. These observations define a connection between induction of lupus-like symptoms and development of severe atherosclerosis in ApoE deficient lupus mouse models.
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http://dx.doi.org/10.1016/j.clim.2008.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2464279PMC
May 2008

Lack of chromatin and nuclear fragmentation in vivo impairs the production of lupus anti-nuclear antibodies.

J Immunol 2007 Dec;179(11):7959-66

Division of Rheumatology, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19140, USA.

Nuclear autoantigens in systemic lupus erythematosus are thought to derive primarily from apoptotic cells, yet there is no direct evidence that interfering with apoptosis impairs the generation of lupus autoantibodies. Here we use a mouse model that lacks the endonuclease caspase-activated DNase (CAD), resulting in an absence of chromatin and nuclear fragmentation during apoptotic cell death. We show that in this mouse, production and release into circulation of chromatin is impaired after exposure to several apoptotic triggers, but that the absence of CAD does not interfere with upstream steps of apoptosis or immune system function. Finally we show that in CAD-mutant mice, impaired lupus autoimmunity is skewed toward known cytoplasmic components, and autoimmunity toward membrane autoantigens is preserved, while autoimmunity toward chromatin and other lupus nuclear targets is severely impaired or absent. We also show, as control, that the induction of experimental autoimmune encephalomyelitis is not affected by the absence of CAD. Thus, our work in vivo strongly suggests that apoptotic molecular steps during cell death generate nuclear autoantigens to sustain the specific autoimmune response in systemic lupus erythematosus.
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http://dx.doi.org/10.4049/jimmunol.179.11.7959DOI Listing
December 2007