Nucleophosmin (NPM1) is an abundant, nucleolar tumor antigen with important roles in cell proliferation and putative contributions to oncogenesis. Wild-type NPM1 forms pentameric oligomers through interactions at the amino-terminal core domain. A truncated form of NPM1 found in some hepatocellular carcinoma tissue formed an unusually stable oligomer and showed increased susceptibility to cleavage by granzyme B. Initiation of translation at the seventh methionine generated a protein (M7-NPM) that shared all these properties. We used deuterium exchange mass spectrometry (DXMS) to perform a detailed structural analysis of wild-type NPM1 and M7-NPM, and found dynamic conformational shifts or local “unfolding” at a specific monomer-monomer interface which included the β-hairpin “latch.” We tested the importance of interactions at the β-hairpin “latch” by replacing a conserved tyrosine in the middle of the β-hairpin loop with glutamic acid, generating Y67E-NPM. Y67E-NPM did not form stable oligomers and further, prevented wild-type NPM1 oligomerization in a dominant-negative fashion, supporting the critical role of the β-hairpin “latch” in monomer-monomer interactions. Also, we show preferential cleavage by granzyme B at one of two available aspartates (either D161 or D122) in M7-NPM and Y67E-NPM, whereas wild-type NPM1 was cleaved at both sites. Thus, we observed a correlation between the propensity to form oligomers and granzyme B cleavage site selection in nucleophosmin proteins, suggesting that a small change at an important monomer-monomer interface can affect conformational shifts and impact protein-protein interactions.
Since dermatomyositis (DM) is associated with an increased risk of malignancy, accurate identification of patients likely to harbor cancers is important. Using immunoprecipitations from radiolabeled cell lysates, several groups recently showed that anti–transcription intermediary factor 1γ (anti–TIF-1γ) antibodies are associated with malignancy in DM. We undertook this study to develop sensitive, specific assays to detect antibodies against TIF-1γ and nuclear matrix protein NXP-2 and to evaluate their association with malignancy in DM.
To detect anti–TIF-1γ antibodies, immunoprecipitations were performed using lysates made from HeLa cells overexpressing TIF-1γ, with detection by immunoblotting. Anti–NXP-2 antibodies were assayed by immunoprecipitation using 35S-methionine–labeled NXP-2 generated by in vitro transcription/translation. We analyzed patient sera from DM cohorts seen at the Stanford University Dermatology Clinic (n = 111) and the Johns Hopkins Myositis Center (n = 102).
A total of 17% and 38% of patients had antibodies against NXP-2 and TIF-1γ, respectively. Reactivity against either NXP-2 or TIF-1γ identified 83% of patients with cancer-associated DM. In addition to older age and male sex, cancer was associated with antibodies to NXP-2 or TIF-1γ on multivariate analysis (odds ratio 3.78 [95% confidence interval 1.33–10.8]). Stratification by sex revealed that anti–NXP-2 was specifically associated with cancer in males (odds ratio 5.78 [95% confidence interval 1.35–24.7]).
These studies demonstrate that anti–NXP-2 and anti–TIF-1γ antibodies are frequent DM specificities (found in 55% of patients) and are present in most patients with cancer-associated DM.
Interferon-alpha (IFNα)–induced thyroid dysfunction occurs in up to 20% of patients undergoing therapy for hepatitis C. The diversity of thyroid disease presentations suggests that several different pathological mechanisms are involved, such as autoimmunity and direct toxicity. Elucidating the relationships between risk factors and disease phenotype provides insight into the mechanisms of disease pathophysiology.
We studied 869 euthyroid patients from the ACHIEVE 2/3 trial, a randomized international clinical trial comparing pegylated-IFNα2a weekly or albumin-IFNα2b every 2 weeks for up to 24 weeks in patients with hepatitis C, genotype 2 or 3, from 136 centers. The study population was 60% male and 55% white. Serum thyrotropin (TSH) and free thyroxine were measured before therapy, monthly during treatment from week 8, and at 4- and 12-week follow-up visits.
Overall, 181 (20.8%) participants had at least one abnormal TSH during the study. Low TSH occurred in 71 (8.2%), of whom 30 (3.5%) had a suppressed TSH below 0.1 mU/L. Hypothyroidism occurred in 53 patients (6.1%), with peak TSH above 10 mU/L in 12 patients (1.4%). Fifty-seven patients had a biphasic thyroiditis (6.6%), with extreme values for the nadir and/or peak TSH in all but one. Medical therapy was given to one thyrotoxic patient, four hypothyroid patients, and 26 biphasic thyroiditis patients. Multivariate logistic regression analysis demonstrated that biphasic thyroiditis is associated with being female and higher pretreatment serum TSH, whereas being Asian or a current smoker decreased the risk of thyroiditis. Hypo- and hyperthyroidism are most strongly predicted by the pretreatment TSH.
Biphasic thyroiditis accounted for the majority (58%) of clinically relevant IFNα-induced thyroid dysfunction. We confirmed our recent findings in a related cohort that female sex is a risk factor for thyroiditis but not hypothyroidism. Further, in this large multiethnic study, the risk of thyroiditis is dramatically increased, specifically for white women. Smoking was found to be protective of thyroiditis. These results support closer monitoring of women and those with a serum TSH at the extremes of the normal range during therapy so that prompt intervention can mitigate the consequences of thyroid dysfunction associated with IFNα treatment.
The objective of this study was to determine whether anti-peptidylarginine deiminase type 4 (PAD4) antibodies were present in first-degree relatives of rheumatoid arthritis (RA) patients in two indigenous North American populations with high prevalence of RA.
Participants were recruited from two indigenous populations in Canada and the United States, including RA patients (probands), their unaffected first-degree relatives, and healthy unrelated controls. Sera were tested for the presence of anti-PAD4 antibodies, anti-cyclic citrullinated peptide (CCP) antibodies, and rheumatoid factor (RF). HLA-DRB1 subtyping was performed and participants were classified according to number of shared epitope alleles present.
Antibodies to PAD4 were detected in 24 of 82 (29.3%) probands; 2 of 147 (1.4%) relatives; and no controls (p <0.0001). Anti-CCP was present in 39/144 (27.1%) of the relatives, and there was no overlap between positivity for anti-CCP and PAD4 in the relatives. In RA patients, anti-PAD4 antibodies were associated with disease duration (p=0.0082) and anti-CCP antibodies (p=0.008), but not smoking or shared epitope alleles.
Despite a significant prevalence of anti-CCP in first-degree relatives, anti-PAD4 antibodies were almost exclusively found in established RA. The prevalence of anti-PAD4 antibodies in RA is similar to the prevalence described in other populations and these autoantibodies are associated with disease duration and anti-CCP in RA.
Arthritis; Rheumatoid; Autoantibodies; peptidylarginine deiminase
A subset of rheumatoid arthritis (RA) patients have detectable antibodies directed against the peptidyl-arginine deiminase (PAD) enzyme isoforms 3 and 4. Anti-PAD3/4 cross-reactive antibodies (anti-PAD3/4XR) have been shown to lower the calcium threshold required for PAD4 activation, an effect potentially relevant to the pathogenesis of RA-associated interstitial lung disease (ILD).
RA patients underwent multi-detector computed tomography (MDCT) of the chest with interpretation by a pulmonary radiologist for ILD features. A semi-quantitative ILD Score (range 0–32) was calculated. Concurrent serum samples were assessed for antibodies against PAD by immunoprecipitation with radiolabeled PAD3 and PAD4.
Among the 176 RA patients studied, any ILD was observed in 58 (33%) and anti-PAD3/4XR was detected in 19 (11%). The frequency of any ILD among those with anti-PAD3/4XR was 68% vs. 29% among those with no anti-PAD (crude OR = 5.39; p = 0.002) and vs. 27% among those with anti-PAD4 that was not cross-reactive with PAD3 (crude OR = 5.74; p = 0.001). Both associations were stronger after adjustment for relevant confounders (adjusted ORs = 7.22 and 6.61, respectively; both p-values<0.01). Among ever smokers with anti-PAD3/4XR, the adjusted frequency of any ILD was 93% vs. 17% for never smokers without the antibody (adjusted OR = 61.4; p = 0.001, p-value for the interaction of smoking with anti-PAD3/4XR<0.05).
The prevalence and extent of ILD was markedly higher among RA patients with anti-PAD3/4 cross-reactive antibodies, even after accounting for relevant confounders, particularly among ever smokers. These findings may suggest etiopathologic mechanisms of RA-ILD, and their clinical utility for predicting ILD warrants additional study.
Autoimmune diseases are thought to be initiated by exposures to foreign antigens that cross-react with endogenous molecules. Scleroderma is an autoimmune connective tissue disease in which patients make antibodies to a limited group of autoantigens, including RPC1, encoded by the POLR3A gene. As patients with scleroderma and antibodies against RPC1 are at increased risk for cancer, we hypothesized that the “foreign” antigens in this autoimmune disease are encoded by somatically mutated genes in the patients’ incipient cancers. Studying cancers from scleroderma patients, we found genetic alterations of the POLR3A locus in six of eight patients with antibodies to RPC1 but not in eight patients without antibodies to RPC1. Analyses of peripheral blood lymphocytes and serum suggested that POLR3A mutations triggered cellular immunity and cross-reactive humoral immune responses. These results offer insight into the pathogenesis of scleroderma and provide support for the idea that acquired immunity helps to control naturally occurring cancers.
Autoantibodies to citrullinated protein antigens are specific markers of rheumatoid arthritis (RA). Although protein citrullination can be activated by numerous stimuli in cells, it remains unclear which of these produce the prominent citrullinated autoantigens targeted in RA. In these studies, we show that RA synovial fluid cells have an unusual pattern of citrullination with marked citrullination of proteins across the broad range of molecular weights, which we term cellular hypercitrullination. Although histone citrullination is a common event during neutrophil activation and death induced by different pathways including apoptosis, NETosis, and necroptosis/autophagy, hypercitrullination is not induced by these stimuli. However, marked hypercitrullination is induced by two immune-mediated membranolytic pathways, mediated by perforin and the membrane attack complex (MAC), which are active in the RA joint and of importance in RA pathogenesis. We further demonstrate that perforin and MAC activity on neutrophils generate the profile of citrullinated autoantigens characteristic of RA. These data suggest that activation of peptidylarginine deiminases during complement and perforin activity may be at the core of citrullinated autoantigen production in RA. These pathways may be amenable to monitoring and therapeutic modulation.
High titer autoantibodies, which are often associated with specific clinical phenotypes, are useful diagnostically and prognostically in systemic autoimmune diseases. In several autoimmune rheumatic diseases (e.g. myositis and Sjogren’s syndrome), 20–40% of patients are autoantibody negative as assessed by conventional assays. The recent discovery of new specificities (e.g., anti-MDA5) in a subset of these autoantibody-negative subjects demonstrates that additional specificities await identification. In this manuscript, we describe a rapid multidimensional method to identify new autoantigens. A central foundation of this rapid approach is the use of an antigen source in which a pathogenic pathway active in the disease is recapitulated. Additionally, the method involves a modified serological proteome analysis strategy which allows confirmation that the correct gel plug has been removed prior to sending for sequencing. Lastly, the approach uses multiple sources of information to enable rapid triangulation and identification of protein candidates. Possible permutations and underlying principles of this triangulation strategy are elaborated to demonstrate the broad utility of this approach for antigen discovery.
Peptidylarginine deiminases (PADs) play a critical role in generating autoantigens in rheumatoid arthritis (RA), but the mechanisms underlying their dysregulation in this disease remain unknown. Although PADs require supraphysiologic concentrations of calcium for activity in vitro, the enzymes are clearly active in vivo (e.g. in RA synovial fluid) where calcium concentrations are much lower. In this study, we have discovered a novel subset of anti-PAD4 autoantibodies (identified by their cross-reactivity with PAD3) which strikingly increase the catalytic efficiency of PAD4 by decreasing the enzyme’s requirement for calcium into the physiologic range. Patients with these novel PAD3/PAD4 cross-reactive autoantibodies had higher baseline radiographic damage scores and a higher likelihood of radiographic progression compared to individuals negative for these antibodies. The ability of autoantibodies to activate an enzyme that itself generates citrullinated autoantigens identifies an important feed-forward loop which may drive the erosive outcome observed in RA patients with these autoantibodies. PAD3 autoantibodies may therefore identify RA patients who would benefit from early aggressive treatment or addition of PAD-inhibitor therapy.
Elevated serum aldolase A levels occur in the absence of elevated creatine kinase M (CK) levels in a subset of myositis patients. This study was undertaken to investigate the cell biology of this unexplained clinical observation.
Cultured human myoblasts were differentiated in vitro. RNA and protein lysates were prepared and used to determine aldolase and CK gene and protein expression by QPCR and immunoblotting. Cardiotoxin was used to induce muscle injury and repair in an experimental mouse model, and aldolase A and CK were immunoblotted in the muscle lysates. Immunohistochemical staining was performed on myositis patient muscle paraffin sections to assess aldolase A and CK staining in vivo.
Aldolase A mRNA and protein expression is highest in differentiating myoblasts, and remains robust throughout differentiation. In contrast, CK mRNA and protein levels are low in undifferentiated myoblasts and become strikingly upregulated as differentiation progresses. Aldolase A protein expression is high in regenerating muscle in the mouse model of injury/repair, while CK expression was low. Immunohistochemical staining of human myositis biopsies showed that muscle cells with the highest levels of aldolase and no CK staining have features of regeneration.
In undifferentiated muscle cells, and those early in the differentiation process, aldolase A is expressed in the absence of CK. Thereafter, both are expressed. We propose that isolated serum aldolase A elevation in myositis patients (i) reflects preferential immune-mediated damage of early regenerative cells, and (ii) is a biomarker of damaged early regenerating muscle cells.
myositis; aldolase; creatine kinase; muscle regeneration
Melanoma differentiation-associated protein 5 (MDA-5) is a novel
autoantibody frequently characterized by interstitial lung disease and a
distinct cutaneous phenotype with palmar papules, ulceration, and rash.
Virtually all patients have underlying dermatomyositis, but many lack the
characteristic clinical myopathy associated with it. In the setting of
amyopathic disease, the absence of clinically available biomarkers or clear
pathologic diagnosis can complicate effective prognostic and therapeutic
intervention. Until recently the presence of MDA-5 antibody associated
dermato-pulmonary syndrome was described only in Asian populations. We present 2
cases of MDA-5-associated dermato-pulmonary syndrome and provide a comprehensive
review of available literature.
A significant body of data implicates the type I interferon (IFN) pathway in the pathogenesis of autoimmune rheumatic diseases. In these disorders, a reinforcing cycle of IFN production can contribute to immunopathology through multiple mechanisms. The type I IFN cytokines are pleiotropic in their effects, mediating anti-viral and anti-tumor activities, and possessing numerous immunomodulatory functions for both the innate and adaptive immune responses. A key principle of the type I IFN system is rapid induction and amplification of the signaling pathway, which generates a feed-forward loop of IFN production, ensuring that a vigorous anti-viral immune response is mounted. While such feed-forward pathways are highly adaptive when it comes to rapid and effective virus eradication, this amplification can be maladaptive in immune responses directed against host tissues. Such feed-forward loops, however, create special opportunities for therapy.
To define the relationship between autoantigen citrullination and different peptidylarginine deiminase (PAD) enzymes in rheumatoid arthritis (RA).
Citrullinated autoantigens were identified by immunoblotting control and ionomycin-activated human primary neutrophil lysate with RA sera. Autoantigen identity and citrullination sites were defined by mass spectrometry. PAD isoenzyme expression in human neutrophils was determined by immunoblotting. PAD substrate specificity was addressed in HL-60 cell lysates co-incubated with human recombinant PAD2, PAD3 and PAD4.
Although prominent protein citrullination is observed in ionomycin activated neutrophils, RA sera only recognized a limited number of these citrullinated molecules. Among these, we identified that beta and gamma actins are citrullinated on at least ten arginine residues, generating a novel 47kDa species that is frequently recognized by RA autoantibodies. Interestingly, we showed that the PAD enzymes expressed in human neutrophils (i.e. PAD2, PAD3 and PAD4) have unique substrate specificities, independent of their subcellular distribution. Thus, only PAD2 was able to citrullinate native beta/gamma-actin, while histone H3 was only citrullinated by PAD4.
These studies identified beta and gamma actins as novel citrullinated autoantigens in RA, allowing enzyme specificity against intracellular substrates to be addressed. The studies provide evidence that PAD enzymes have the intrinsic capacity to select unique protein targets. We propose that unique PAD specificity may play a role in autoantigen selection in RA.
Citrullination; rheumatoid arthritis; anti-CCP; actin; peptidylarginine deiminase
Glycosylation represents an important modification that regulates biological processes in tissues relevant for disease pathogenesis in systemic sclerosis (SSc), including the endothelium and extracellular matrix. Whether patients with systemic sclerosis (SSc) develop antibodies to carbohydrates is not known.
To determine the prevalence and clinical phenotype associated with serum IgG antibodies recognizing distinct glycans in patients with systemic sclerosis (SSc).
Pooled sera from patients with SSc and controls were screened for the presence of specific anti-carbohydrate antibodies using a novel array containing over 300 glycans. Antibody titers to 4-sulfated N-Acetyl-lactosamine (4S-LacNAc, [4OSO3]Galβ1-4GlcNAc) were determined in 181 individual sera from SSc patients by ELISA and associated with disease phenotype.
4S-LacNAc was identified as a target in pooled SSc serum. Anti-4SLAcNAc antibodies were detected in 27/181 (14.9%) of SSc patients compared to 1/40 (2.5%) of healthy controls. Sulfation at position C-4 of galactose (4S-LacNAc) was found to be critical for immunogenicity. Anti-4SLacNAc antibody positive SSc patients had a higher prevalence of pulmonary hypertension by echocardiography (15/27; 55.7% versus anti-4S LacNac negative patients 49/154; 31.8% p=0.02) with an odds ratio of 2.6 (CI 1.1, 6.3). Anti-4S-LacNAc positive patients accounted for 23.4% of all patients with pulmonary hypertension.
Sera from SSc patients contain IgG antibodies targeting distinct sulfated carbohydrates. The presence of anti-4S-LacNAc antibodies is associated with a high prevalence of pulmonary hypertension. These results suggest that specific posttranslational carbohydrate modifications may act as important immunogens in SSc and may contribute to disease pathogenesis.
Scleroderma; carbohydrates; antibodies; pulmonary hypertension
Tissue-infiltrating multinucleated giant cells (MNGs) within geographic necrosis are pathologic hallmarks of granulomatosis with polyangiitis (GPA). However, the origin, phenotype, and function of these cells in GPA remain undefined.
MNG phenotype in GPA lung tissue was examined by immunohistochemistry using antibody directed against cathepsin K and calcitonin-receptor. Tartrate-resistant-acid-phosphatase (TRAP) expression was assessed using enzymatic color reaction. Peripheral blood mononuclear cells (PBMCs) from 13 GPA patients (5 with localized and 8 with systemic disease) and 11 healthy controls were cultured in the presence of RANKL and M-CSF for 9 days, and TRAP+ MNGs containing 3 or more nuclei were identified. GPA lung granulomata contained numerous MNGs that expressed osteoclastic TRAP and cathepsin K but not calcitonin receptors. In the presence of RANKL and M-CSF, PBMCs of GPA patients formed significantly more MNGs than healthy controls (114±29 MNG/well vs. 22±9 MNG/well, P = 0.02). In a subgroup analysis, patients with systemic disease generated significantly more MNGs than patients with localized disease (161±35 MNG/well vs. 39±27 MNG/well, P<0.01) or healthy controls (P<0.01). MNG production did not differ between localized GPA and control subjects (P = 0.96).
MNGs in granulomata in the GPA lung express osteoclastic enzymes TRAP and cathepsin K. GPA patients have a higher propensity to form TRAP+ MNGs from peripheral blood than healthy controls. These data suggest that (i) the tendency to form MNGs is a component of the GPA phenotype itself, and (ii) that lesional MNGs might participate in the destructive process through their proteolytic enzymes.
Dermatomyositis (DM) is a multisystem autoimmune disease, in which serologic evidence of immune responses to disease-specific antigenic targets is found in approximately 50% to 70% of patients. Recently, melanoma differentiation-associated gene 5 (MDA5) has been identified as a DM-specific autoantigen that appears to be targeted in patients with DM and mild or absent muscle inflammation and with an increased risk of interstitial lung disease.
We wished to understand the role of MDA5 in DM skin inflammation by testing it to determine if a specific cutaneous phenotype is associated with MDA5 reactivity.
We retrospectively screened plasma from 77 patients with DM in the outpatient clinics at the Stanford University Department of Dermatology in California.
We found that 10 (13%) patients had circulating anti-MDA5 antibodies, and had a characteristic cutaneous phenotype consisting of skin ulceration, tender palmar papules, or both. Typical areas of skin ulceration included the lateral nailfolds, Gottron papules, and elbows. Biopsy specimens of the palmar papules showed a vasculopathy characterized by vascular fibrin deposition with variable perivascular inflammation. Patients with anti-MDA5 antibodies also had an increased risk of oral pain and/or ulceration, hand swelling, arthritis/arthralgia, and diffuse hair loss. Consistent with previous reports, these patients had little or no myositis and had increased risk of interstitial lung disease.
This study was conducted at a tertiary referral center. Multiple associations with MDA5 antibodies were tested retrospectively on a relatively small cohort of 10 anti-MDA5-positive patients.
We suggest that MDA5 reactivity in DM characterizes a patient population with severe vasculopathy.
autoantibodies; clinically amyopathic dermatomyositis antibody; 140 kd (CADM-140) peptide; dermatomyositis; human; interferon-induced helicase 1 protein; interstitial; lung diseases; phenotype; ulcer
Purpose of review
Most epidemiologic studies have demonstrated an increased risk of cancer in scleroderma patients. Reasons for this risk increase have been poorly understood and often attributed to cytotoxic therapies or damage from scleroderma. Recognition that some patients have a close temporal relationship between cancer diagnosis and scleroderma clinical onset has focused attention on the possibility that scleroderma may be a paraneoplastic syndrome in a subset of patients. This review will discuss the latest epidemiologic data linking cancer and scleroderma and explore a model for the development of paraneoplastic scleroderma.
New investigations have demonstrated an association between RNA polymerase III autoantibodies and a close temporal relationship between cancer diagnosis and the development of clinical scleroderma. A unique nucleolar RNA polymerase III expression pattern has been identified in malignant tissue from these scleroderma patients suggesting that autoantigen expression in the cancer and the autoantibody response are associated. Similar data in inflammatory myositis have illustrated that disease-specific autoantigens may be expressed in cancers and damaged target tissues (muscle) undergoing regeneration.
These data suggest a model of paraneoplastic autoimmunity in which cross-reactive immune responses may target autoantigens that are expressed in both cancers and diseased autoimmune target tissues.
autoantibodies; cancer; paraneoplastic; systemic sclerosis
In addition to inducing a self-limited myopathy, statin use is associated with an immune-mediated necrotizing (IMNM) myopathy with autoantibodies recognizing ~ 200 and ~100 kDa autoantigens. Identifying these molecules will clarify disease mechanism and facilitate diagnosis.
The effect of statin treatment on autoantigen expression was addressed by immunoprecipitation using patient sera. The identity of the ~100 kDa autoantigen was confirmed by immunoprecipitating in vitro-translated HMGCR protein. HMGCR expression in muscle was analyzed by immunofluorescence. A cohort of myopathy patients was screened for anti-HMGCR autoantibodies by ELISA and genotyped for the rs4149056 C allele, a predictor of self-limited statin myopathy.
Statin exposure induced expression of the ~200/~100 kDa autoantigens in cultured cells. HMGCR was identified as the ~100 kDa autoantigen. Competition experiments demonstrated no distinct autoantibodies recognizing the ~200 kDa protein. In muscle biopsies from anti-HMGCR positive patients, HMGCR expression was up-regulated in cells expressing NCAM, a marker of muscle regeneration. Anti-HMGCR autoantibodies were found in 45 of 750 patients presenting to the Johns Hopkins Myositis Center (6%). Among patients age 50 or older, 92% were exposed to statins. The prevalence of the rs4149056 C allele was not increased in anti-HMGCR subjects.
Statins up-regulate expression of HMGCR, the major target of autoantibodies in statin-associated IMNM. Regenerating muscle cells express high levels of HMGCR, which may sustain the immune response even after statins are discontinued. These studies demonstrate a mechanistic link between an environmental trigger and the development of sustained autoimmunity. Detection of anti-HMGCR autoantibodies may facilitate diagnosis and direct therapy.
The aim of this study was to explore the presence and localization of myocardial citrullination in samples from rheumatoid arthritis (RA) patients compared to rheumatic and non-rheumatic disease control groups.
Archived myocardial samples obtained during autopsy from 1995 to 2009 were assembled into four groups: RA; scleroderma; fatal myocarditis; and non-rheumatic disease controls. Samples were examined by immunohistochemistry (IHC) for the presence and localization of citrullination and peptidyl arginine deiminase enzymes (PADs) by a single cardiovascular pathologist blinded to disease group and clinical characteristics.
Myocardial samples from seventeen RA patients were compared with those from fourteen controls, five fatal myocarditis patients, and ten scleroderma patients. Strong citrullination staining was detected exclusively in the myocardial interstitium in each of the groups. However, average and peak anti-citrulline staining was 59% and 44% higher, respectively, for the RA group compared to the combined non-RA groups (P < 0.05 for both comparisons). Myocardial fibrosis did not differ between the groups. In contrast to citrullination, PADs 1 to 3 and 6 were detected in cardiomyocytes (primarily PADs 1 and 3), resident inflammatory cells (primarily PADs 2 and 4), and, to a smaller extent, in endothelial cells and vascular smooth muscle cells. PAD staining did not co-localize with anti-citrulline staining in the interstitium and did not vary by disease state.
Staining for citrullination was higher in the myocardial interstitium of RA compared to other disease states, a finding that could link autoimmunity to the known increase in myocardial dysfunction and heart failure in RA.
Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a function in the brain has been well established in vivo, and in vitro studies have shown that its activity is essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2 alternative splice forms of UFD2a, UFD2a-7 and -7/7a, are expressed sequentially during myoblast differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal muscle. In vertebrates (humans, rodents, zebrafish), UFD2a-7/7a is expressed only in mature striated muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific splicing regulatory motifs located in the 3′ introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most likely imparts divergent functions that are important for myogenisis.
To determine if antibodies against peptidyl arginine deiminase Type 4 (PAD-4) are present in the pre-clinical phase of rheumatoid arthritis (RA), and to compare their appearance to other pre-clinical autoantibodies.
Prediagnosis serum samples from 83 subjects with RA were evaluated for presence of anti-PAD-4 antibody, anti-cyclic citrullinated peptide (anti-CCP) antibody, and rheumatoid factor (RF). In addition, a control cohort (N = 83) matched on age, gender, race, number of samples, and duration of serum storage were tested for antibody against PAD-4 to determine its sensitivity and specificity for future RA.
Fifteen of 83 (18.1%) subjects with RA had at least one prediagnosis sample positive for anti-PAD-4. One of 83 (1.2%) control subjects had at least one sample positive, resulting in a sensitivity and specificity of anti-PAD-4 for the future development of RA of 18.1% and 98.8%, respectively. The mean time of first positivity for anti-PAD-4 was ~4.6 years prior to diagnosis. Anti-PAD-4 positivity was associated with anti-CCP positivity (OR 5.13, 95%CI 1.07–24.5, p = 0.04). In subjects with prediagnosis samples positive for both antibodies, anti-CCP positivity predated anti-PAD-4 in 9 of 13 (69%) cases.
Autoantibodies to PAD-4 are present in the pre-clinical phase of RA in a subset of patients and are associated with anti-CCP positivity. Further exploration is needed regarding the timing of appearance and disease-related effects of PAD-4 autoimmunity.
Rheumatoid arthritis; pre-clinical; peptidyl arginine deiminase type 4; anti-citrullinated peptide antibodies
We examined the temporal relationship between scleroderma development and malignancy, and evaluated whether this differed by autoantibody status among affected patients.
Participants had a diagnosis of scleroderma, cancer, an available serum sample, and a cancer pathology specimen. Sera were tested for autoantibodies against topoisomerase I, centromere, and RNA polymerase I/III by immunoprecipitation and/or ELISA. Clinical and demographic characteristics were compared across autoantibody categories. Expression of RNA polymerases I and III was evaluated by immunohistochemistry using cancerous tissue from patients with anti-RNA polymerase antibodies.
Twenty three subjects were enrolled. Six subjects tested positive for anti-RNA polymerase I/III (Pol), 5 for anti-topoisomerase I (Topo), 8 for anti-centromere (CENP), and 4 recognized none of these antigens (Negative). The median duration of scleroderma at cancer diagnosis differed significantly between groups: −1.2 years (Pol), +13.4 years (Topo), +11.1 years (CENP), and +2.3 years (Negative) (p=0.027). RNA polymerase III demonstrated a robust nucleolar staining pattern in 4 of 5 available tumors from patients with antibodies to RNA polymerase I/III. In contrast, nucleolar RNA polymerase III staining was not detected in any of 4 examined tumors in the RNA polymerase antibody-negative group (p=0.048).
There is a close temporal relationship between onset of cancer and scleroderma in patients with antibodies to RNA polymerase I/III, which is distinct from scleroderma patients with other autoantibody specificities. In this study, autoantibody response and tumor antigen expression are associated. We propose that malignancy may initiate the scleroderma-specific immune response and drive disease in a subset of scleroderma patients.
The systemic autoimmune diseases are a complex group of disorders characterized by elaboration of high titer autoantibodies and immune-mediated damage of tissues. Two striking features of autoimmune rheumatic diseases are their self-sustaining nature and capacity for auto-amplification, exemplified by disease flares. These features suggest the presence of a feed-forward cycle in disease propagation, in which immune effector pathways drive the generation/release of autoantigens, which in turn fuel the immune response. There is a growing awareness that structural modification during cytotoxic granule-induced cell death is a frequent and striking feature of autoantigens, and may be an important principle driving disease. This review focuses on granzyme B (GrB)-mediated cleavage of autoantigens including (i) features of GrB cleavage sites within autoantigens, (ii) co-location of cleavage sites with autoimmune epitopes, and (iii) GrB-sensitivity of autoantigens in disease-relevant target tissue. The mechanisms whereby GrB-induced changes in autoantigen structure may contribute to the initiation and propagation of autoimmunity are reviewed and reveal that GrB has the potential to create or destroy autoimmune epitopes. As there remains no direct evidence demonstrating a causal role for GrB-cleavage of antigens in the generation of autoimmunity, this review highlights important outstanding questions about the role of GrB in autoantigen selection.
granzyme; autoimmunity; autoantibody; autoantigen; antigen processing; proteolysis
Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS resulting from a progressive loss of oligodendrocytes. Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes of the brain, and postmortem sections show concurrent loss of myelin basic protein and TAL from sites of demyelination. Infiltrating CD8+ CTLs are thought to play a key role in oligodendrocyte cell death. Cleavage by granzyme B (GrB) is predictive for autoantigenicity of self-proteins, thereby further implicating CTL-induced death in the initiation and propagation of autoimmunity. The precursor frequency and CTL activity of HLA-A2–restricted TAL 168–176–specific CD8+ T cells is increased in MS patients. In this paper, we show that TAL, but not myelin basic protein, is specifically cleaved by human GrB. The recognition site of GrB that resulted in the cleavage of a dominant TAL fragment was mapped to a VVAD motif at aa residue 27 by N-terminal sequencing and confirmed by site-directed mutagenesis. The major C-terminal GrB cleavage product, residues 28–337, had no enzymatic activity but retained the antigenicity of full-length TAL, effectively stimulating the proliferation and CTL activity of PBMCs and of CD8+ T cell lines from patients with MS. Sera of MS patients exhibited similar binding affinity to wild-type and GrB-cleaved TAL. Because GrB mediates the killing of target cells and cleavage by GrB is predictive of autoantigen status of self proteins, GrB-cleaved TAL-specific T cell-mediated cytotoxicity may contribute to the progressive destruction of oligodendrocytes in patients with MS.
To address mechanisms that control the activity of human peptidyl arginine deiminase type 4 (PAD-4).
PAD-4 autocitrullination was determined by anti–modified citrulline immunoblotting, using purified recombinant and endogenous PAD-4 from activated human primary neutrophils and cell lines expressing PAD-4. The citrullination sites in PAD-4 were determined by mass spectrometry. Mechanisms of autocitrullination-induced inactivation and the functional consequences of autocitrullination in PAD-4 polymorphic variants were addressed using purified components and cell lines expressing PAD-4 wild-type, PAD-4 mutant, and PAD-4 polymorphic variants relevant to rheumatoid arthritis (RA).
PAD-4 is autocitrullinated in vitro and during activation of primary cells and cell lines expressing PAD-4. Interestingly, this modification inactivated the function of the enzyme. The efficiency of inactivation differed among genetically defined PAD-4 variants relevant to RA. PAD-4 was citrullinated at 10 sites, which are clustered into 3 distinct regions, including a cluster of arginines around the active site cleft where Arg-372 and -374 were identified as the potential autocitrullination targets that inactivate the enzyme. Autocitrullination also modified the structure of PAD-4, abrogating its recognition by multiple rabbit antibodies, but augmenting its recognition by human anti–PAD-4 autoantibodies.
Our findings suggest that autocitrullination regulates the production of citrullinated proteins during cell activation, and that this is affected by structural polymorphisms in PAD-4. Autocitrullination also influences PAD-4 structure and immune response.