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Antony Rosen

Publications and source records attributed to Antony Rosen.

At least 19 recordsLinked to original sources
PubMed2007

Apoptotic splenocytes drive the autoimmune response to poly(ADP-ribose) polymerase 1 in a murine model of lupus.

Although defects in apoptosis have been linked to both human and murine lupus, the exact mechanisms remain unknown. Moreover, it is not clear whether such defects are primary or secondary events in disease pathogenesis. To address these issues, we used an induced model of murine lupus, the parent-into-F(1) model of chronic graft-versus-host disease (cGVHD) in which a lupus-like phenotype highly similar to human systemic lupus erythematosus is reliably induced in normal F(1) mice. We addressed the role of nuclear Ags modified by caspases during apoptosis as potential targets of the autoantibody response and our results identify poly(ADP-ribose) polymerase 1 (PARP-1) as a frequently targeted autoantigen. Additional proteins cleaved during apoptosis were also targeted by the immune response. Importantly, female mice exhibited significantly greater numbers of apoptotic cells in germinal centers and higher serum anti-PARP-1 Ab levels compared with male cGVHD mice. Serum anti-PARP-1 levels in male cGVHD mice could be elevated to levels comparable to those of female cGVHD mice by the injection of apoptotic syngeneic F(1) splenocytes early in the disease course. These results provide a mechanism by which lupus autoantibodies target apoptotic molecules. Specifically, T cell-driven polyclonal B cell activation characteristic of systemic lupus erythematosus is sufficient to saturate otherwise normal apoptotic clearance mechanisms, permitting apoptotic material to accumulate, serve as autoantigens, and drive autoantibody production.

Animals↗
PubMed2006

Mouse and human granzyme B have distinct tetrapeptide specificities and abilities to recruit the bid pathway.

Granzyme B is an important mediator of cytotoxic lymphocyte granule-induced death of target cells, accomplishing this through cleavage of Bid and cleavage and activation of caspases as well as direct cleavage of downstream substrates. Significant controversy exists regarding the primary pathways used by granzyme B to induce cell death, perhaps arising from the use of different protease/substrate combinations in different studies. The primary sequence of human, rat, and mouse granzymes B is well conserved, and the substrate specificity and crystal structure of the human and rat proteases are extremely similar. Although little is known about the substrate specificity of mouse granzyme B, recent studies suggest that it may differ significantly from the human protease. In these studies we show that the specificities of human and mouse granzymes B differ significantly. Human and mouse granzyme B cleave species-specific procaspase-3 more efficiently than the unmatched substrates. The distinct specificities of human and mouse granzyme B highlight a previously unappreciated requirement for Asp(192) in the acquisition of catalytic activity upon cleavage of procaspase-3 at Asp(175). Although human granzyme B efficiently cleaves human or mouse Bid, these substrates are highly resistant to cleavage by the mouse protease, strongly indicating that the Bid pathway is not a major primary mediator of the effects of mouse granzyme B. These studies provide important insights into the substrate specificity and function of the granzyme B pathway in different species and highlight that caution is essential when designing and interpreting experiments with different forms of granzyme B.

Animals↗
PubMed2006

The MPAC domain is a novel mitotically regulated domain, removed by apoptotic protease cleavage during cell death.

The apoptotic proteases, including caspases and granzyme B, have independent evolutionary origins, yet are both highly specific for cleavage after aspartic acid residues and cleave many of the same substrates at closely spaced sites. In addition, many of these substrates are also reversibly regulated during other processes such as the cell cycle. In these studies, we have identified a novel domain (the MPAC domain: Mitotically Phosphorylated, Apoptotically Cleaved) present at the N-terminus of Ufd2a, which is regulated both by cleavage during cell death, and by phosphorylation during mitosis. We have also identified a corresponding domain, at the C-terminus of polyA polymerase (PAP), which is similarly regulated by phosphorylation during mitosis and is delineated by an apoptotic protease cleavage site. The positioning of the apoptotic cleavage site suggests that it represents a novel connector between the regulatory domain and its functional partner(s), providing insights into the structure and function that guided the evolution of the apoptotic proteases.

Apoptosis↗
PubMed2006

Granzyme B mediates neurotoxicity through a G-protein-coupled receptor.

Neuroinflammatory diseases such as multiple sclerosis (MS) are characterized by focal regions of demyelination and axonal loss associated with infiltrating T cells. However, the role of activated T cells in causing neuronal injury remains unclear. CD4 and CD8 T cells were isolated from normal donors and polyclonally activated using plate-bound anti-CD3 and soluble anti-CD28. The conditioned T cell supernatants caused toxicity to cultured human fetal neurons, which could be blocked by immunodepleting the supernatants of granzyme B (GrB). Recombinant GrB also caused toxicity in neurons by caspase-dependent pathways but no toxicity was seen in astrocytes. The neurotoxicity was independent of perforin and could not be blocked by mannose-6-phosphate. However, GrB-induced neurotoxicity was sensitive to pertussis toxin, implicating the stimulation of Gialpha protein-coupled receptors. GrB caused a decrease in cAMP levels but only modest increases in intracellular calcium. The effect on intracellular calcium could be markedly potentiated by stromal-derived factor 1alpha. GrB-induced neurotoxicity could also be blocked by vitamin E and a neuroimmunophilin ligand. In conclusion, GrB may be an important mediator of neuronal injury in T cell-mediated neuroinflammatory disorders.

Antioxidants↗
PubMed2006

Sexual dimorphism in immune response genes as a function of puberty.

BACKGROUND: Autoimmune diseases are more prevalent in females than in males, whereas males have higher mortality associated with infectious diseases. To increase our understanding of this sexual dimorphism in the immune system, we sought to identify and characterize inherent differences in immune response programs in the spleens of male and female mice before, during and after puberty. RESULTS: After the onset of puberty, female mice showed a higher expression of adaptive immune response genes, while males had a higher expression of innate immune genes. This result suggested a requirement for sex hormones. Using in vivo and in vitro assays in normal and mutant mouse strains, we found that reverse signaling through FasL was directly influenced by estrogen, with downstream consequences of increased CD8+ T cell-derived B cell help (via cytokines) and enhanced immunoglobulin production. CONCLUSION: These results demonstrate that sexual dimorphism in innate and adaptive immune genes is dependent on puberty. This study also revealed that estrogen influences immunoglobulin levels in post-pubertal female mice via the Fas-FasL pathway.

Animals↗
PubMed2005

Apoptosis and autoimmunity.

Autoimmune diseases reflect the confluence of genetic, environmental and stochastic events. Recent studies have implicated apoptotic cell death pathways in initiating and propagating autoimmune diseases, as well as in rendering individuals susceptible to such diseases. Similar to autoimmunity, apoptosis is a multistep process, affecting immune and target cells, integrating numerous intrinsic and extrinsic signals, and requiring the actions of multiple gene products. Particularly relevant to the complexity of autoimmunity are the recent observations that apoptotic death might provide a primary source of tolerogen to shape the immune repertoire, or be the target of the immune response in autoimmunity, and that apoptosis is both required for lymphocyte selection and immunoregulation, and is a prominent outcome of immune and inflammatory effector pathways.

Animals↗
PubMed2005

Caspase-resistant Golgin-160 disrupts apoptosis induced by secretory pathway stress and ligation of death receptors.

Golgin-160 is a coiled-coil protein on the cytoplasmic face of the Golgi complex that is cleaved by caspases during apoptosis. We assessed the sensitivity of cell lines stably expressing wild-type or caspase-resistant golgin-160 to several proapoptotic stimuli. Cells expressing a caspase-resistant mutant of golgin-160 were strikingly resistant to apoptosis induced by ligation of death receptors and by drugs that induce endoplasmic reticulum (ER) stress, including brefeldin-A, dithiothreitol, and thapsigargin. However, both cell lines responded similarly to other proapoptotic stimuli, including staurosporine, anisomycin, and etoposide. The caspase-resistant golgin-160 dominantly prevented cleavage of endogenous golgin-160 after ligation of death receptors or induction of ER stress, which could be explained by a failure of initiator caspase activation. The block in apoptosis in cells expressing caspase-resistant golgin-160 could not be bypassed by expression of potential caspase cleavage fragments of golgin-160, or by drug-induced disassembly of the Golgi complex. Our results suggest that some apoptotic signals (including those initiated by death receptors and ER stress) are sensed and integrated at Golgi membranes and that golgin-160 plays an important role in transduction of these signals.

Anisomycin↗
PubMed2005

Enhanced autoantigen expression in regenerating muscle cells in idiopathic inflammatory myopathy.

Unique autoantibody specificities are strongly associated with distinct clinical phenotypes, making autoantibodies useful for diagnosis and prognosis. To investigate the mechanisms underlying this striking association, we examined autoantigen expression in normal muscle and in muscle from patients with autoimmune myositis. Although myositis autoantigens are expressed at very low levels in control muscle, they are found at high levels in myositis muscle. Furthermore, increased autoantigen expression correlates with differentiation state, such that myositis autoantigen expression is increased in cells that have features of regenerating muscle cells. Consistent with this, we found that cultured myoblasts express high levels of autoantigens, which are strikingly down-regulated as cells differentiate into myotubes in vitro. These data strongly implicate regenerating muscle cells rather than mature myotubes as the source of ongoing antigen supply in autoimmune myositis. Myositis autoantigen expression is also markedly increased in several cancers known to be associated with autoimmune myositis, but not in their related normal tissues, demonstrating that tumor cells and undifferentiated myoblasts are antigenically similar. We propose that in cancer-associated myositis, an autoimmune response directed against cancer cross-reacts with regenerating muscle cells, enabling a feed-forward loop of tissue damage and antigen selection. Regulating pathways of antigen expression may provide unrecognized therapeutic opportunities in autoimmune diseases.

Autoantigens↗
PubMed2005

Generation of novel covalent RNA-protein complexes in cells by ultraviolet B irradiation: implications for autoimmunity.

OBJECTIVE: To determine whether ultraviolet B (UVB) irradiation induces novel modifications in autoantigens targeted during experimental photoinduced epidermal damage. METHODS: To search for novel UVB-induced autoantigen modifications, lysates made from UVB-irradiated human keratinocytes or HeLa cells were immunoblotted using human autoantibodies that recognize ribonucleoprotein autoantigens. Novel autoantigen structures identified were further characterized using nucleases and RNA hybridization. RESULTS: Human sera that recognize U1-70 kd (U1-70K) and La by immunoblotting also recognized multiple novel species when they were used to immunoblot lysates of UVB-irradiated keratinocytes or HeLa cells. These species were not present in control cells and were not observed when apoptosis was induced by Fas ligation or cytotoxic lymphocyte granule contents. Biochemical analysis using multiple assays revealed that these novel UVB-induced molecular species result from the covalent crosslinking between the U1 RNA and the hYRNA molecules with their associated proteins, including U1-70K, La, and likely components of the Sm particle. CONCLUSION: These data demonstrate that UVB irradiation of live cells can directly induce covalent RNA-protein complexes, which are recognized by human autoantibodies. As previously described for other autoantigens, these covalent complexes of RNA and proteins may have important consequences in terms of antigen capture and processing.

Apoptosis↗
PubMed2005

Activation of the endoplasmic reticulum stress response in autoimmune myositis: potential role in muscle fiber damage and dysfunction.

OBJECTIVE: The etiology and pathogenesis of human inflammatory myopathies remain unclear. Findings of several studies suggest that the degree of inflammation does not correlate consistently with the severity of clinical disease or of structural changes in the muscle fibers, indicating that nonimmune pathways may contribute to the pathogenesis of myositis. This study was undertaken to investigate these pathways in myositis patients and in a class I major histocompatibility complex (MHC)-transgenic mouse model of myositis. METHODS: We examined muscle tissue from human myositis patients and from class I MHC-transgenic mice for nonimmune pathways, using biochemical, immunohistochemical, and gene expression profiling assays. RESULTS: Up-regulation of class I MHC in skeletal muscle fibers was an early and consistent feature of human inflammatory myopathies. Class I MHC staining in muscle fibers of myositis patients showed both cell surface and a reticular pattern of internal reactivity. The pathways of endoplasmic reticulum (ER) stress response, the unfolded protein response (glucose-regulated protein 78 pathway), and the ER overload response (NF-kappaB pathway) were significantly activated in muscle tissue of human myositis patients and in the mouse model. Ectopic expression of wild-type mouse class I MHC (H-2K(b)) but not degradable glycosylation mutants of H-2K(b) induced ER stress response in C(2)C(12) skeletal muscle cells. CONCLUSION: These results indicate that the ER stress response may be a major nonimmune mechanism responsible for skeletal muscle damage and dysfunction in autoimmune myositis. Strategies to interfere with this pathway may have therapeutic value in patients with this disease.

Animals↗
PubMed2005

Autoantigens: the critical partner in initiating and propagating systemic autoimmunity.

The increasing recognition that cancer is frequently associated with an autoantibody response, and observations that systemic autoimmunity is sometimes associated with the diagnosis of a variety of malignancies (many detected near the onset of autoimmune disease), strongly underscore a potential mechanistic connection between cancer immunity and systemic autoimmunity. Accumulating data suggest that autoantigens are critical partners in driving the autoimmune response. Furthermore, unique changes in antigen expression and conformation in the immunizing tumor and the target tissue may play a role in antigen selection and ongoing damage. This construct has important implications for diagnosis, monitoring, and treatment of autoimmunity and, potentially, cancer immunotherapy.

Autoantigens↗
PubMed2005

Evaluation of topoisomerase-1-specific CD8+ T-cell response in systemic sclerosis.

Measurement of disease activity in systemic autoimmune disorders is often unreliable, and immunosuppressive therapy is often titrated to crude clinical response and/or onset of complications. Systemic sclerosis (SSc) presents a distinct clinical phenotype associated with specific autoantibodies. Anti-topoisomerase-1 (SCL-70) is selectively detected in 30-60% of subjects with diffuse skin and interstitial lung involvement. Such patients offer an ideal clinical model to characterize and quantify the autoantigen-specific T-cell response and its correlation with disease phenotype and activity. Human leukocyte antigen A2 (HLA-A2)-restricted topo-1 peptides were selected based on an epitope prediction algorithm. For initial studies, the best binder topo-1(262-270) KMLDHEYTT (#262) was used alone or loaded onto an artificial antigen-presenting platform generated by coupling a dimeric major histocompatibility complex-immunoglobulin G fusion protein (HLA-A2-Ig) and anti-CD28 antibodies onto magnetic beads (artificial antigen-presenting cells). Blood samples (100 microL) from HLA-A2+ SSc patients and cytomegalovirus (CMV) seropositive healthy control subjects were tested in an intracellular cytokine staining assay. Gamma interferon production by CD8+ T cells was measured after stimulation with peptide #262, CMVpp65, or MART-1 (irrelevant peptide). In two of five SCL-70+ patients, peptide #262-loaded aAPCs induced a specific CD8+ T-cell response (0.45% +/- 0.23% of total CD8+ cells). This response was not observed in the seven SCL-70- (five SSc and two CMV+) control subjects studied (0.03% +/- 0.02%). Interestingly, bronchoalveolar lavage fluid obtained from one topo-1-responsive SSc patient who had worsening respiratory function and active alveolitis showed striking enrichment of topo-1-specific CD8+ T cells (3.94%). This small-volume ex vivo assay may prove to be a sensitive and specific tool to assess disease activity and to monitor response to therapy in patients with scleroderma.

Autoimmune Diseases↗
PubMed2004

Ufd2, a novel autoantigen in scleroderma, regulates sister chromatid separation.

The characterization of molecules recognized by patients with autoimmune diseases has provided significant insights into important biological pathways. In these studies, we define Ufd2 (a novel E4 polyubiquitylating enzyme) as a new autoantigen in scleroderma, and show that it regulates chromosome condensation and separation during mitosis in human cells. Ufd2 is regulated by phosphorylation in mitosis. Inhibition of Ufd2 expression results in mitotic arrest at the metaphase-anaphase transition, where cells manifest abnormal chromosome morphology, missegregated chromosomes, irregular spindles, and premature separation of sister chromatids. This is accompanied by premature separase activation, and accumulation of securin in a novel modified form. We further demonstrate that Ufd2 directly and efficiently ubiquitylates securin in vitro and is required for securin polyubiquitylation in vivo. This is the first description of a physiologic substrate for Ufd2, establishing this E4 enzyme as an important regulator of chromosome condensation and separation during mitosis in human cells. Its targeting in scleroderma, together with many other components of the mitotic machinery, reinforces the concept that mitotic cells may be an important focus of the autoimmune response in this disease.

Apoptosis↗
PubMed2004

Selective cleavage of nucleolar autoantigen B23 by granzyme B in differentiated vascular smooth muscle cells: insights into the association of specific autoantibodies with distinct disease phenotypes.

OBJECTIVE: To investigate the association of specific autoantibodies with distinct disease phenotypes. The association of autoantibodies to nucleophosmin/B23 with pulmonary hypertension in scleroderma, and the susceptibility of autoantigens to cleavage by granzyme B (GB), provided a focus for these studies. METHODS: Intact cells were subjected to cytotoxic lymphocyte granule-induced death, and the susceptibility of autoantigens to cleavage by GB was addressed by immunoblotting and/or by a novel immunofluorescence assay. RESULTS: B23 was cleaved efficiently by GB in vitro, but was highly resistant to cleavage by GB during cytotoxic lymphocyte granule-mediated death of many intact cell types. In contrast, this molecule was highly susceptible to GB-mediated proteolysis exclusively in differentiated vascular smooth muscle cells. Topoisomerase I and several other GB substrates did not show this striking change in cleavage susceptibility in different cell types. CONCLUSION: These data demonstrate that the cleavage of B23 by GB in intact cells is dependent upon both cell type and phenotype. The susceptibility of this autoantigen (which is associated with a distinct pulmonary vascular phenotype in scleroderma) to GB-mediated proteolysis selectively in vascular smooth muscle cells suggests that the GB-cleavable conformation of autoantigens may occur selectively in the target tissue, and may play a role in shaping the phenotype-specific autoimmune response.

Autoantigens↗
PubMed2004

Altered structure of autoantigens during apoptosis.

The clustering and concentration of autoantigens at the surface of apoptotic cells, in combination with the striking tolerance-inducing function of apoptotic cells, have focused attention on abnormalities in apoptotic cell execution and clearance as potential susceptibility and initiating factors in systemic autoimmunity. Structural changes that occur during cell death may influence the immunogenicity of self antigens. This article discusses the modifications that autoantigens undergo during cell death, identifies certain proimmune forms of apoptotic death in which autoantigen structure is frequently modified, and reviews the mechanisms through which such structural changes might lead to initiation of an autoimmune response.

Apoptosis↗
PubMed2004

Granzyme B-induced cell death.

Granzyme B (GrB) is a serine protease that is released by cytotoxic lymphocytes to kill virus-infected and tumor cells. Recent advances in the understanding of GrB have stressed the importance of reassessing the mechanisms by which GrB accomplishes its death functions. These include the uptake and trafficking of GrB within target cells, pathways used to trigger cell death, and the mechanism(s) controlling its killing activity. In addition, the role that GrB plays in human pathologies is still to be defined. The purpose of this review is to discuss recent insights into the biology of GrB and to evaluate its functional significance in health and disease.

Apoptosis↗
PubMed2004

Methods for inducing apoptosis.

Apoptotic cells are sources of tolerogenic material during tissue homeostasis; abnormalities in apoptosis or in the clearance of apoptotic material generate a novel source of antigens against which an autoimmune response may be initiated. In our laboratory, we study the biochemistry and cell biology of systemic autoimmune disease autoantigens during different forms of cell death. Several different methods for inducing apoptosis, and for assaying the induction of this cellular process, are routinely performed. This chapter describes methods for inducing apoptosis via ultraviolet B irradiation, small molecule drug treatments, death receptor ligation, and exposure to granule components of cytotoxic lymphocytes. Assays to confirm the induction of apoptosis by quantifying changes in mitochondrial membrane potential, phosphatidylserine membrane localization, DNA content, and autoantigen cleavage are also detailed.

Annexin A5↗