Anti-MDA5 positive clinically amyopathic dermatomyositis presenting with severe cardiomyopathy.

BACKGROUND: Anti-MDA5 (Melanoma differentiation-associated gene 5) positive dermatomyositis is a new variant of clinically amyopathic dermatomyositis that presents with characteristic mucocutaneous findings and is associated with a higher risk of developing rapidly progressive interstitial lung disease. Because its presentation differs from that of classical dermatomyositis, this entity can be a diagnostic challenge for the clinician. METHODS & RESULTS: We present the case of a 55-year-old male with a 7-month history of chill sensation, constitutional symptoms and polyarthralgia. Within 3 months, the patient developed progressive heart failure with dyspnoea and orthopnoea, together with characteristic cutaneous lesions. Skin biopsies demonstrated thrombosis of small and medium-sized arteries in the reticular dermis, together with an evolved lobular panniculitis and prominent mucin deposits. CONCLUSIONS: Clinicians should be aware of the characteristic clinical and histopathologic presentation of this variant of dermatomyositis to establish an early diagnosis. Further evidence is needed to clarify the risk of cardiac involvement in this subset of patients.

PM-Scl and Th/To in systemic sclerosis: a comparison of different autoantibody assays.

OBJECTIVE: To compare test characteristics of the Euroimmun line blot assay with other assays for two uncommon autoantibody specificities in systemic sclerosis (SSc). METHODS: Patients from the Johns Hopkins Scleroderma Center were assayed routinely using the Euroimmun platform. Patients positive for anti-Th/To (N = 73) and anti-PM-Scl (PM75 and/or PM100; N = 290) by Euroimmun were compared with SSc patients negative for these autoantibodies. For Th/To antibodies, the comparison assay was immunoprecipitation (IP), performed using 4 Th/To complex components: POP1, RPP40, RPP30, and RPP25. For anti-PM-Scl, IPs were performed with PM100 and PM75. Different Euroimmun cut-offs for assigning antibody positive status (≥ 15/+, ≥ 36/++, ≥ 71/+++) were examined. Kappa statistics were calculated to determine agreement between assays. RESULTS: The best performing thresholds for defining anti-PM-Scl positivity were both PM75 and PM100 ≥ 15/+ on Euroimmun, corresponding to a kappa statistic of 0.79, sensitivity 72% and specificity 100%. For anti-Th/To, kappa values were lower for all comparisons (κ < 0.5). Given the high sensitivity of defining anti-Th/To by ≥ 15/+ (91-95%), a potential approach is to use Euroimmun screening (15/+ cut-off), followed by confirmatory IP. CONCLUSION: Given the increasing utilization of Euroimmun and the importance of comparing data across cohorts, continued use of this platform is warranted, acknowledging discordance with IP for some specificities. For these, using a two-step approach (Euroimmun to maximize sensitivity, confirmatory assay to increase specificity) is suggested. KEY POINTS: • For less common SSc autoantibody specificities, some discordances exist between IP and Euroimmun LIA. • The best performing thresholds for defining anti-PM-Scl positivity were both PM75 and PM100 ≥ 15/+ on Euroimmun. • For Th/To, a two-step approach (Euroimmun to maximize sensitivity, confirmatory assay to increase specificity) is suggested.

Novel packages of viral and self-antigens are generated during apoptosis.

Immune context is an essential determinant of the host response to potential autoantigens. The clustering of the autoantigens targeted in systemic lupus erythematosus within surface blebs of apoptotic cells generates high concentrations of autoantigen within discrete subcellular packages. We demonstrate here that when apoptosis is induced by Sindbis virus infection, viral antigens and autoantigens cocluster exclusively in small surface blebs of apoptotic cells. The surface of these blebs is rich in viral glycoproteins, and virions can be seen blebbing from their surface. We propose that these blebs of mixed foreign and self-origin define a novel immune context that may challenge self-tolerance.

Scleroderma autoantigens are uniquely fragmented by metal-catalyzed oxidation reactions: implications for pathogenesis.

The observation that revelation of immunocryptic epitopes in self antigens may initiate the autoimmune response has prompted the search for processes which induce novel fragmentation of autoantigens as potential initiators of autoimmunity. The reversible ischemia reperfusion which characterizes scleroderma has focused attention on reactive oxygen species as molecules which might induce autoantigen fragmentation. We demonstrate that several of the autoantigens targeted in diffuse scleroderma are uniquely susceptible to cleavage by reactive oxygen species, in a metal-dependent manner. Multiple features of the fragmentation reaction and its inhibition indicate that these autoantigens possess metal-binding sites, which focus metal-catalyzed oxidation reactions (and consequent fragmentation) to specific regions of the antigens. These data suggest that the autoantibody response in scleroderma is the immune marker of unique protein fragmentation, induced by ischemia reperfusion in the presence of appropriate metals, and focus attention on abnormal metal status as a potential pathogenic principle in this disease.

Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes.

Systemic lupus erythematosus is a multisystem autoimmune disease in which the autoantibody response targets a variety of autoantigens of diverse subcellular location. We show here that these autoantigens are clustered in two distinct populations of blebs at the surface of apoptotic cells. The population of smaller blebs contains fragmented endoplasmic reticulum (ER) and ribosomes, as well as the ribonucleoprotein, Ro. The larger blebs (apoptotic bodies) contain nucleosomal DNA, Ro, La, and the small nuclear ribonucleoproteins. These autoantigen clusters have in common their proximity to the ER and nuclear membranes, sites of increased generation of reactive oxygen species in apoptotic cells. Oxidative modification at these sites may be a mechanism that unites this diverse group of molecules together as autoantigens.

DNA-dependent protein kinase is one of a subset of autoantigens specifically cleaved early during apoptosis.

Proteolytic cleavage of key substrates appears to be an important biochemical mechanism underlying the apoptotic process, and the centrality of interleukin 1 beta-converting enzyme (ICE)-like proteases as mediators of apoptosis has been suggested. The identification of the relevant substrates of the ICE protease family during apoptosis therefore constitutes a major challenge. Using human autoantibodies, we demonstrate here that a subset of autoantigens is specifically cleaved early during apoptosis. One of these cleaved molecules is identified as the catalytic subunit of the DNA-dependent protein kinase. The time courses of all proteolytic cleavages are identical and coincide with the onset of morphologic apoptosis. Furthermore, all cleavages share the same inhibition characteristics, which implicate an ICE-like activity(ies). We propose that cleavage of these autoantigens targets these molecules for an autoimmune response by revealing immunocryptic fragments in a proimmune apoptotic setting. Study of the immunogenicity of these fragments may yield insights into the autoimmune targeting of molecules. Moreover, the autoantibodies described will be valuable tools for the elucidation of mechanistically important proteolytic steps along the apoptotic pathway.

Cleavage by granzyme B is strongly predictive of autoantigen status: implications for initiation of autoimmunity.

Systemic autoimmune diseases are a genetically complex, heterogeneous group of disorders in which the immune system targets a diverse but highly specific group of intracellular autoantigens. The molecules targeted are not unified by common structure, function, or distribution in control cells but become clustered and concentrated in surface blebs when cells undergo apoptosis. We show here that the majority of autoantigens targeted across the spectrum of human systemic autoimmune diseases are efficiently cleaved by granzyme B in vitro and during cytotoxic lymphocyte granule-induced death, generating unique fragments not observed during any other form of apoptosis. These molecules are not cleaved by caspase-8, although this protease has a very similar specificity to granzyme B. The granzyme B cleavage sites in autoantigens contain amino acids in the P(2) and P(3) positions that are preferred by granzyme B but are not tolerated by caspase-8. In contrast to autoantigens, nonautoantigens are either not cleaved by granzyme B or are cleaved to generate fragments identical to those formed in other forms of apoptosis. The striking ability of granzyme B to generate unique fragments is therefore an exclusive property of autoantigens and unifies the majority of molecules targeted in this spectrum of diseases. These results focus attention on the role of the cytotoxic lymphocyte granule-induced death pathway in the initiation and propagation of systemic autoimmunity.

Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death.

Proteolysis mediated by the interleukin 1 beta-converting enzyme (ICE) homologues is an important mechanism of the apoptotic process. The ICE homologue apopain/CPP-32/Yama (subsequently referred to as apopain) cleaves poly(ADP-ribose)polymerase (PARP) early during apoptosis. Additional apoptosis-specific protein cleavages have been observed in which the direct involvement of ICE-like proteases has been postulated. These substrates include the 70-kD protein component of the U1-ribonucleoprotein (U1-70kD), and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). The present studies demonstrate that U1-70kD and DNA-PKcs are excellent substrates for apopain, with cleavage occurring at sites that are highly similar to the cleavage site within PARP. The fragments generated from isolated protein substrates by apopain are identical to those observed in intact apoptotic cells, in apoptotic cell extracts, and in normal cell extracts to which apopain has been added. Like PARP, cleavage of these substrates in apoptotic cell extracts is abolished by nanomolar concentrations of Ac-DEVD-CHO and micromolar amounts of Ac-YVAD-CHO, confirming the involvement of apopain or an apopain-like activity. We propose that a central function of apopain or similar homologues in apoptosis is the cleavage of nuclear repair proteins, thereby abolishing their critical homeostatic functions.

Autoantigens in systemic autoimmunity: critical partner in pathogenesis.

Rosen A, Casciola-Rosen L (Johns Hopkins University School of Medicine, Baltimore, MD, USA). Autoantigens in systemic autoimmunity: critical partner in pathogenesis (Review). J Intern Med 2009; 265: 625-631.Understanding the mechanisms of human autoimmune rheumatic diseases presents a major challenge, due to marked complexity involving multiple domains, including genetics, environment and kinetics. In spite of this, the immune response in each of these diseases is largely specific, with distinct autoantibodies associated with different disease phenotypes. Defining the basis of such specificity will provide important insights into disease mechanism. Accumulating data suggest an interesting paradigm for antigen selection in autoimmunity, in which target tissue and immune effector pathways form a mutually reinforcing partnership. In this model, distinct autoantibody patterns in autoimmunity may be viewed as the integrated, amplified output of several interacting systems, including: (i) the specific target tissue, (ii) the immune effector pathways that modify antigen structure and cause tissue damage and dysfunction, and (iii) the homeostatic pathways activated in response to damage (e.g. regeneration/differentiation/cytokine effects). As unique antigen expression and structure may occur exclusively under these amplifying circumstances, it is useful to view the molecules targeted as 'neo-antigens', that is, antigens expressed under specific conditions, rather than ubiquitously. This model adds an important new dynamic element to selection of antigen targets in autoimmunity, and suggests that the amplifying loop will only be identified by studying the diseased target tissue in vivo.

Caspase-2 is localized at the Golgi complex and cleaves golgin-160 during apoptosis.

Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors.

The caspase-3 precursor has a cytosolic and mitochondrial distribution: implications for apoptotic signaling.

Caspase-3-mediated proteolysis is a critical element of the apoptotic process. Recent studies have demonstrated a central role for mitochondrial proteins (e.g., Bcl-2 and cytochrome c) in the activation of caspase-3, by a process that involves interaction of several protein molecules. Using antibodies that specifically recognize the precursor form of caspase-3, we demonstrate that the caspase-3 proenzyme has a mitochondrial and cytosolic distribution in nonapoptotic cells. The mitochondrial caspase-3 precursor is contained in the intermembrane space. Delivery of a variety of apoptotic stimuli is accompanied by loss of mitochondrial caspase-3 precursor staining and appearance of caspase-3 proteolytic activity. We propose that the mitochondrial subpopulation of caspase-3 precursor molecules is coupled to a distinct subset of apoptotic signaling pathways that are Bcl-2 sensitive and that are transduced through multiple mitochondrion-specific protein interactions.

Granzyme B: evidence for a role in the origin of myasthenia gravis.

PURPOSE OF RESEARCH: Although the pathogenesis of myasthenia gravis (MG) as an antibody mediated disorder of acetylcholine receptors (AChRs) at neuromuscular junctions is well understood, the origin of the autoimmune response is unclear. The thymus is intimately involved in initiation of the autoimmune response; the antigen, AChR, is present in the thymus, but how the autoimmune response is triggered is not known. Granzyme B (GrB), a proteolytic enzyme present in cytolytic T cells and natural killer (NK) cells, selectively cleaves many potential autoantigens (but few non-autoantigens), generating novel fragments that trigger autoreactive responses. This protease has been strongly implicated in the pathogenesis of several autoimmune diseases including lupus, rheumatoid arthritis, dermatomyositis, and others. In the studies described in this manuscript, we examined the ability of GrB to cleave the AChR subunits, and performed biochemical, immunohistochemical and molecular studies on thymus glands from myasthenic patients and controls to assess GrB expression. MAIN RESULTS: GrB efficiently and specifically cleaves subunits of AChR, especially the epsilon subunit. GrB is present in thymus glands from myasthenia patients, but is absent in control thymuses. CONCLUSIONS: Our results provide evidence supporting a potential role for GrB in the process of initiation of MG, and are consistent with the concept of an immunodominant epsilon epitope.

Bcl-2-dependent oxidation of pyruvate dehydrogenase-E2, a primary biliary cirrhosis autoantigen, during apoptosis.

The close association between autoantibodies against pyruvate dehydrogenase-E2 (PDC-E2), a ubiquitous mitochondrial protein, and primary biliary cirrhosis (PBC) is unexplained. Many autoantigens are selectively modified during apoptosis, which has focused attention on apoptotic cells as a potential source of "neo-antigens" responsible for activating autoreactive lymphocytes. Since increased apoptosis of bile duct epithelial cells (cholangiocytes) is evident in patients with PBC, we evaluated the effect of apoptosis on PDC-E2. Autoantibody recognition of PDC-E2 by immunofluorescence persisted in apoptotic cholangiocytes and appeared unchanged by immunoblot analysis. PDC-E2 was neither cleaved by caspases nor concentrated into surface blebs in apoptotic cells. In other cell types, autoantibody recognition of PDC-E2, as assessed by immunofluorescence, was abrogated after apoptosis, although expression levels of PDC-E2 appeared unchanged when examined by immunoblot analysis. Both overexpression of Bcl-2 and depletion of glutathione before inducing apoptosis prevented this loss of autoantibody recognition, suggesting that glutathiolation, rather than degradation or loss, of PDC-E2 was responsible for the loss of immunofluorescence signal. We postulate that apoptotic cholangiocytes, unlike other apoptotic cell types, are a potential source of immunogenic PDC-E2 in patients with PBC.

Relationship Between Neuromyelitis Optica Spectrum Disorder and Sjögren's Syndrome: Central Nervous System Extraglandular Disease or Unrelated, Co-Occurring Autoimmunity?

OBJECTIVE: Sjögren's syndrome (SS) patients may be affected by the neuromyelitis optica spectrum disorder (NMOSD), a severe demyelinating syndrome associated with anti-aquaporin 4 antibodies (anti-AQP-4 antibodies). The relationship between SS and NMOSD has been a sustained focus of investigation. Among SS patients, anti-AQP-4 antibodies have been detected exclusively in those with NMOSD. It has therefore been speculated that NMOSD is not a neurologic complication of SS. However, such studies evaluated small numbers of SS patients, often mixed with other inflammatory disorders. METHODS: We compared frequencies of anti-AQP-4 and SS-associated antibodies in 109 SS patients, including 11 with NMOSD, 8 with non-NMOSD demyelinating syndromes, and 90 without demyelinating syndromes. RESULTS: When assessed using a fluorescence-activated cell sorting (FACS) assay, anti-AQP-4 antibodies were seen exclusively in those SS patients with NMOSD (72.7%), but not in SS patients without NMOSD (P < 0.01). In contrast, anti-Ro 52, anti-Ro 60, and other autoantibodies were not more prevalent in SS patients with NMOSD versus those without. Anti-AQP-4 antibodies were detected more frequently among NMOSD patients by FACS assay than with a commercial immunohistochemical assay (72.7% versus 54.5%), despite assessment after a more prolonged period of immunosuppressive therapy (median 38 months versus 5 months; P < 0.01). CONCLUSION: The syndrome-specificity of anti-AQP-4 antibodies, along with an otherwise similar antibody profile in SS NMOSD patients, indicates that NMOSD is not a direct central nervous system manifestation of SS. Anti-AQP-4 antibodies can persist and be refractory to prolonged immunosuppressive therapy.

Autoantigens as substrates for apoptotic proteases: implications for the pathogenesis of systemic autoimmune disease.

Systemic autoimmune diseases are a genetically complex, heterogeneous group of diseases in which the immune system targets a diverse, but highly specific group of intracellular autoantigens. The clustering and marked concentration of these molecules in the surface blebs of apoptotic cells, and their modification by apoptosis-specific proteolytic cleavage and/or phosphorylation at these sites, has focused attention on a unique apoptotic setting as the potential initiating stimulus for systemic autoimmunity. This apoptotic event is likely to (i) occur in a microenvironment containing high concentrations of the targeted antigens, (ii) be pro-immune in nature (e.g. viral infection), and (iii) allow suprathreshold concentrations of antigen with non-tolerized structure (either novel fragments, post-translational modifications, or complexes) to enter the class II processing pathway and initiate a primary immune response. Defective clearance or reduced anti-inflammatory consequences of apoptotic material may be important susceptibility factors in this group of diseases. Once the primary immune response to apoptotic antigens has been initiated, other apoptotic events (occurring in the course of homeostasis or damage) may stimulate the secondary immune response with less stringency, resulting in flares.

Sequential activation of three distinct ICE-like activities in Fas-ligated Jurkat cells.

ICE family proteases have been implicated as important effectors of the apoptotic pathway, perhaps acting hierarchically in a protease cascade. Using cleavage of endogenous protease substrates as probes, three distinct tiers of ICE-like activity were observed after Fas ligation in Jurkat cells. The earliest cleavage detected (30 min) was of fodrin, and produced a 150 kDa fragment. The second phase of cleavage (50 min) involved PARP, U1-70kDa and DNA-PKcs, all substrates of the CPP32-like proteases. Lamin B cleavage was observed during the third cleavage phase (90 min). Distinct inhibition profiles obtained using a panel of peptide-based inhibitors of ICE-like proteases clearly distinguished the three different cleavage phases. These studies provide evidence for a sequence of ICE-like proteolytic activity during apoptosis. The early fodrin cleavage, producing a 150 kDa fragment, identifies an ICE-like activity proximal to CPP32 in Fas-induced Jurkat cell apoptosis.

Caspase-mediated proteolysis during apoptosis: insights from apoptotic neutrophils.

Apoptosis is initiated by activation of caspases (interleukin 1beta-converting enzyme homologues), which cause coordinated cleavage of several death substrates that function in structural or homeostatic pathways. The relationship between substrate cleavage and apoptosis is not yet known, nor is it clear whether cleavage of specific substrates is a critical requirement for apoptosis. The human neutrophil provides novel insights into the roles of proteolysis of specific substrates during apoptosis, since only a subset of caspase substrates are present in mature neutrophils. Of the death substrates we screened, PARP, the nuclear mitotic apparatus protein (NuMA), the 70 kDa subunit of the U1 small ribonucleoprotein (U1-70kDa) and the catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) were not detected in non-apoptotic neutrophils; in contrast, lamin B and fodrin were present in amounts similar to those found in other cells. Caspase-3 activity was absent in freshly isolated neutrophils, but was detected when neutrophils were aged in vitro, coincident with the onset of morphologic and biochemical apoptosis. The absence of PARP, NuMA, U1-70kDa and DNA-PK(CS) in non-apoptotic neutrophils suggests that these are not critical anti-apoptotic proteins, and that their fragments are not required components of the neutrophil apoptotic pathway. These studies highlight the conserved role of caspase activation in the apoptotic mechanism, and focus attention on several conserved structural substrates as potential transducers of the proteolytic signal in apoptosis.

Apoptosis in systemic lupus erythematosus. Clinical implications.

SLE is a heterogeneous and complex group of disorders of uncertain cause. Recent studies have suggested that abnormalities in the apoptotic cell death process may play an important role in the initiation and propagation of this spectrum of disease by altering the generation and cleavage of antigens, and through abnormalities in immunoregulation. The clustering and concentration of autoantigens in and on the surface blebs of apoptotic cells, modifications of antigen structure during certain forms of apoptotic death, and abnormalities in apoptotic cell clearance in humans with SLE and in certain animal models are reviewed and synthesized into a comprehensive model of systemic autoimmunity.

Clinical and pathologic differences in interstitial lung disease based on antisynthetase antibody type.

BACKGROUND: Interstitial lung disease (ILD) is a common extramuscular manifestation of the idiopathic inflammatory myopathies (IIMs), dermatomyositis (DM) and polymyositis (PM). Patients with antisynthetase antibodies (ASA) demonstrate some or all of the features of the antisynthetase syndrome including IIM and ILD. It has been hypothesized that the clinical expression of antisynthetase syndrome varies between specific ASAs. OBJECTIVE: We sought to determine whether the myositis-associated ILD (MA-ILD) phenotype differs based on the presence of ASAs and by ASA subtype. METHODS: A cross-sectional and longitudinal analysis of consecutive patients enrolled at the Johns Hopkins Myositis Center with ILD in the setting of clinically diagnosed autoimmune myositis was conducted. RESULTS: Seventy-seven subjects were included; 36 were ASA negative, 28 were anti-Jo1 positive, and 13 were non-Jo1 ASA positive (5 anti-PL-12, 4 anti-PL-7, 2 anti-EJ, and 2 anti-OJ). Non-Jo1 ASA positive participants were more likely to be African-American than Caucasian as compared to both the anti-Jo1 positive (p = 0.01) and ASA negative groups (p < 0.01). ASA negative participants had better mean forced vital capacity percent predicted (FVC%) and total computed tomography scores over time compared to those with anti-Jo1 after controlling for potential confounders. CONCLUSIONS: ASA status was significantly different by race. Those with anti-Jo1 antibodies had worse lung function and CT scores over time compared to those without detectable antisynthetase antibodies. Further prospective study in a larger cohort is needed to determine whether these apparent antibody-specific differences in demographics and manifestations of disease translate into meaningful disparities in clinical outcomes.