ESTIMATING AUTOANTIBODY SIGNATURES TO DETECT AUTOIMMUNE DISEASE PATIENT SUBSETS

Autoimmune diseases are characterized by highly specific immune responses against molecules in self-tissues. Different autoimmune diseases are characterized by distinct immune responses, making autoantibodies useful for diagnosis and prediction. In many diseases, the targets of autoantibodies are incompletely defined. Although the technologies for autoantibody discovery have advanced dramatically over the past decade, each of these techniques generates hundreds of possibilities, which are onerous and expensive to validate. We set out to establish a method to greatly simplify autoantibody discovery, using a pre-filtering step to define subgroups with similar specificities based on migration of labeled, immunoprecipitated proteins on sodium dodecyl sulfate (SDS) gels and autoradiography [Gel Electrophoresis and band detection on Autoradiograms (GEA)]. Human recognition of patterns is not optimal when the patterns are complex or scattered across many samples. Multiple sources of errors - including irrelevant intensity differences and warping of gels - have challenged automation of pattern discovery from autoradiograms. In this paper, we address these limitations using a Bayesian hierarchical model with shrinkage priors for pattern alignment and spatial dewarping. The Bayesian model combines information from multiple gel sets and corrects spatial warping for coherent estimation of autoantibody signatures defined by presence or absence of a grid of landmark proteins. We show the preprocessing creates better separated clusters and improves the accuracy of autoantibody subset detection via hierarchical clustering. Finally, we demonstrate the utility of the proposed methods with GEA data from scleroderma patients

A Hierarchical Role for Classical Pathway Complement Proteins in the Clearance of Apoptotic Cells in Vivo

The strongest susceptibility genes for the development of systemic lupus erythematosus (SLE) in humans are null mutants of classical pathway complement proteins. There is a hierarchy of disease susceptibility and severity according to the position of the missing protein in the activation pathway, with the severest disease associated with C1q deficiency. Here we demonstrate, using novel in vivo models of apoptotic cell clearance during sterile peritonitis, a similar hierarchical role for classical pathway complement proteins in vivo in the clearance of apoptotic cells by macrophages. Our results constitute the first demonstration of an impairment in the phagocytosis of apoptotic cells by macrophages in vivo in a mammalian system. Apoptotic cells are thought to be a major source of the autoantigens of SLE, and impairment of their removal by complement may explain the link between hereditary complement deficiency and the development of SLE

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

Histidyl–tRNA Synthetase and Asparaginyl–tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells

Autoantibodies to histidyl–tRNA synthetase (HisRS) or to alanyl–, asparaginyl–, glycyl–, isoleucyl–, or threonyl–tRNA synthetase occur in ∼25% of patients with polymyositis or dermatomyositis. We tested the ability of several aminoacyl–tRNA synthetases to induce leukocyte migration. HisRS induced CD4+ and CD8+ lymphocytes, interleukin (IL)-2–activated monocytes, and immature dendritic cells (iDCs) to migrate, but not neutrophils, mature DCs, or unstimulated monocytes. An NH2-terminal domain, 1–48 HisRS, was chemotactic for lymphocytes and activated monocytes, whereas a deletion mutant, HisRS-M, was inactive. HisRS selectively activated CC chemokine receptor (CCR)5-transfected HEK-293 cells, inducing migration by interacting with extracellular domain three. Furthermore, monoclonal anti-CCR5 blocked HisRS-induced chemotaxis and conversely, HisRS blocked anti-CCR5 binding. Asparaginyl–tRNA synthetase induced migration of lymphocytes, activated monocytes, iDCs, and CCR3-transfected HEK-293 cells. Seryl–tRNA synthetase induced migration of CCR3-transfected cells but not iDCs. Nonautoantigenic aspartyl–tRNA and lysyl–tRNA synthetases were not chemotactic. Thus, autoantigenic aminoacyl–tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses. Therefore, the selection of a self-molecule as a target for an autoantibody response may be a consequence of the proinflammatory properties of the molecule itself

Ordering the Cytochrome c–initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9–dependent Manner

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1–mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c–inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9