Autoantibodies against B23, a nucleolar phosphoprotein,occur in scleroderma and are associated with pulmonary hypertension

Objective

To determine whether the abundant nucleolar phosphoprotein B23 is a target of autoantibodies in scleroderma, and to examine the clinical phenotype associated with these antibodies.

Methods

Ninety‐two randomly selected scleroderma sera were screened by enzyme‐linked immunosorbent assay against recombinant human B23. Demographic, clinical, and serologic parameters associated with B23 autoantibody status were examined.

Results

We demonstrated that autoantibodies against B23 occur in ～11% of sera obtained from patients with scleroderma. B23 seropositivity was related to pulmonary hypertension, antifibrillarin antibody, anti‐RNP antibody, and decreased lung capacity. In multivariate analysis, B23 autoantibodies remained strongly associated with moderate‐to‐severe pulmonary hypertension and antifibrillarin antibodies.

Conclusion

These data unite B23 with the group of nucleolar autoantigens targeted in scleroderma and thus focus attention on changes in the nucleolus that render its components immunogenic in this disease. The demonstration that antibodies to B23 are associated with an increased prevalence of pulmonary hypertension points to anti‐B23 antibodies as a possible marker of a specific phenotype in scleroderma.

     

Lack of evidence of stimulatory autoantibodies to platelet‐derived growth factor receptor in patients with systemic sclerosis

Objective

Systemic sclerosis (SSc) is a severe connective tissue disease of unknown etiology, characterized by fibrosis of the skin and multiple internal organs. Recent findings suggested that the disease is driven by stimulatory autoantibodies to platelet‐derived growth factor receptor (PDGFR), which stimulate the production of reactive oxygen species (ROS) and collagen by fibroblasts. These results opened novel avenues of research into the diagnosis and treatment of SSc. The present study was undertaken to confirm the presence of anti‐PDGFR antibodies in patients with SSc.

Methods

Immunoglobulins from 37 patients with SSc were purified by protein A/G chromatography. PDGFR activation was tested using 4 different sensitive bioassays, i.e., cell proliferation, ROS production, signal transduction, and receptor phosphorylation; the latter was also tested in a separate population of 7 patients with SSc from a different research center.

Results

Purified IgG samples from patients with SSc were positive when tested for antinuclear autoantibodies, but did not specifically activate PDGFRα or PDGFRβ in any of the tests. Cell stimulation with PDGF itself consistently produced a strong signal.

Conclusion

The present results raise questions regarding the existence of agonistic autoantibodies to PDGFR in SSc.

     

Anti‐DNA topoisomerase IIα autoantibodies in localized scleroderma

Objective

To determine the prevalence and clinical correlation of anti‐DNA topoisomerase IIα (anti–topo IIα) antibody in patients with localized scleroderma.

Methods

Anti–topo IIα antibodies or anti‐DNA topoisomerase I (topo I) antibodies were determined by enzyme‐linked immunosorbent assay (ELISA) and immunoblotting. Inhibition of topo IIα enzymatic activity by the antibodies was evaluated by decatenation assays using kinetoplast DNA as a substrate.

Results

IgG or IgM anti–topo IIα antibody was detected in 76% (35 of 46) of patients with localized scleroderma, and in 85% (11 of 13) of patients with generalized morphea, the severest form of localized scleroderma. This prevalence of the antibody in patients with localized scleroderma was much higher than that found in patients with systemic sclerosis (SSc) (5 of 37 [14%]), systemic lupus erythematosus (2 of 26 [8%]), dermatomyositis (2 of 20 [10%]), and in healthy controls (3 of 42 [7%]). Immunoblotting confirmed the presence of IgG anti–topo IIα antibody in sera from patients with localized scleroderma and showed no cross‐reactivity of anti–topo IIα antibody with topo I. Anti–topo I antibody was not detected by ELISA in any sera from patients with localized scleroderma. In addition, anti–topo I antibody from SSc patients did not cross‐react with topo IIα. The presence of anti–topo IIα antibody was associated with a greater total number of sclerotic lesions and number of plaque lesions in patients with localized scleroderma. Furthermore, anti–topo IIα antibody was able to inhibit topo IIα enzymatic activity.

Conclusion

The results of the present study indicate that anti–topo IIα is a major autoantibody in localized scleroderma, and is distinct from anti–topo I antibody in SSc.

     

A possible mechanism for endogenous activation of the type I interferon system in myositis patients with anti–Jo‐1 or anti–Ro 52/anti–Ro 60 autoantibodies

Objective

To investigate type I interferon (IFN) system activation and its correlation with autoantibodies and organ manifestations in polymyositis (PM), dermatomyositis (DM), and inclusion body myositis.

Methods

Sera from 30 patients and 16 healthy controls, or purified IgG, were combined with material released from necrotized cells to stimulate IFNα production by peripheral blood mononuclear cells (PBMCs) from healthy blood donors. Muscle biopsy specimens from 25 patients and 7 healthy controls were investigated for blood dendritic cell antigen 2 (BDCA‐2)–positive plasmacytoid dendritic cells (PDCs) and IFNα/β‐inducible myxovirus resistance 1 (MX‐1) protein.

Results

Sera from 13 patients who were positive for anti–Jo‐1 or anti–Ro 52/anti–Ro 60 autoantibodies induced IFNα production in PBMCs when combined with necrotic cell material. In addition, IgG prepared from anti–Jo‐1–positive PM sera induced IFNα with necrotic material, but not when the latter was treated with RNase. BDCA‐2 expression in PDCs in muscle tissue was increased in PM patients with anti–Jo‐1 autoantibodies, while MX‐1 staining in capillaries was increased in DM patients, compared with healthy individuals. IFNα‐inducing capacity correlated with interstitial lung disease, while MX‐1 expression in the capillaries correlated with DM.

Conclusion

Immune complexes containing anti–Jo‐1 or anti–Ro 52/anti–Ro 60 autoantibodies and RNA may act as endogenous IFNα inducers that activate IFNα production in PDCs. These PDCs could be of importance for inducing myositis, whereas in DM patients without autoantibodies the presence of MX‐1 protein in capillaries suggests another cellular IFNα source and induction mechanism. Consequently, the type I IFN system may be of importance in both PM and DM, but via different pathways.

     

PM–Scl‐75 is the main autoantigen in patients with the polymyositis/scleroderma overlap syndrome

Objective

To compare the autoantigenicity of the recently described N‐terminally elongated PM–Scl‐75 protein with that of PM–Scl‐100 and the originally defined PM–Scl‐75 polypeptide, and to determine its value for analyzing sera from patients with the polymyositis (PM)/scleroderma overlap syndrome.

Methods

Serum samples obtained from patients with the PM/scleroderma overlap syndrome and from patients with several other diseases were analyzed for the presence of autoantibodies reactive with recombinant PM–Scl‐100 and PM–Scl‐75 (both the original and the longer form) proteins, in an enzyme‐linked immunosorbent assay (ELISA).

Results

Autoantibodies recognizing the longer PM–Scl‐75 protein isoform were detected in 28% of the patients with PM/scleroderma. This percentage is slightly higher than that for PM–Scl‐100 (25%) and is significantly higher than that for the previously defined PM–Scl‐75 protein (11%). In addition, we identified a significant number of patients who had anti–PM–Scl‐75 but not anti–PM–Scl‐100 antibodies. This finding contrasts with what has been previously reported for the shorter version of the PM–Scl‐75 protein.

Conclusion

Our data indicate that use of the long PM–Scl‐75 isoform in addition to PM–Scl‐100 in ELISAs significantly increases the number of patients in whom anti–PM‐Scl autoantibodies can be detected.

     

C1D is a major autoantibody target in patients with the polymyositis–scleroderma overlap syndrome

Objective

To assess whether the recently discovered exosome‐associated proteins MPP6, C1D, KIAA0052/hMtr4, hSki2, and hSki8 are targeted by autoantibodies, and to determine whether these autoantibodies are accompanied by antibodies directed to the established exosome‐associated autoantigens PM–Scl‐75 and PM–Scl‐100.

Methods

Complementary DNAs encoding the recently identified human exosome–associated proteins were expressed as His‐tagged fusion proteins in Escherichia coli cells. Sera obtained from patients with several different autoimmune diseases were analyzed for the presence of autoantibodies directed to these proteins, in an enzyme‐linked immunosorbent assay (ELISA). The ELISA data obtained for C1D were confirmed by Western blot analysis, using recombinant C1D.

Results

All exosome‐associated proteins were found to be targeted by autoantibodies, although the frequency with which such antibodies occurred in patient sera was relatively low, with the exception of anti‐C1D antibodies. Autoantibodies recognizing C1D were detected in 7 of 30 patients (23%) with the polymyositis (PM)–scleroderma overlap syndrome; this frequency was similar to the frequencies for the established autoantigens PM–Scl‐75c (27%) and PM–Scl‐100 (23%). Importantly, several patients with the PM–scleroderma overlap syndrome had anti‐C1D antibodies but no anti–PM‐Scl antibodies. Anti‐C1D autoantibodies were observed in only 2 of 204 patients with other diseases, including PM, dermatomyositis, and scleroderma.

Conclusion

Our results demonstrate that the recently identified exosome‐associated protein C1D is a major autoantigen in patients with the PM–scleroderma overlap syndrome and suggest that the use of recombinant C1D as an autoantibody target may aid in diagnosis of the PM–scleroderma overlap syndrome.

     

Coordinate overexpression of interferon‐α–induced genes in systemic lupus erythematosus

Objective

To study the contribution of interferon‐α (IFNα) and IFNγ to the IFN gene expression signature that has been observed in microarray screens of peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE).

Methods

Quantitative real‐time polymerase chain reaction analysis of healthy control PBMCs was used to determine the relative induction of a panel of IFN‐inducible genes (IFIGs) by IFNα and IFNγ. PBMCs from 77 SLE patients were compared with those from 22 disease controls and 28 healthy donors for expression of IFIGs.

Results

Expression of IFNα‐inducible genes was significantly higher in SLE PBMCs than in those from disease controls or healthy donors. The level of expression of all IFIGs in PBMCs from SLE patients with IFNα pathway activation correlated highly with the inherent responsiveness of those genes to IFNα, suggesting coordinate activation of that cytokine pathway. Expression of genes preferentially induced by IFNγ was not significantly increased in SLE PBMCs compared with control PBMCs. IFNα‐regulated gene‐inducing activity was detected in some SLE plasma samples.

Conclusion

The coordinate activation of IFNα‐induced genes is a characteristic of PBMCs from many SLE patients, supporting the hypothesis that IFNα is the predominant stimulus for IFIG expression in lupus.

     

Induction of interferon‐α production in plasmacytoid dendritic cells by immune complexes containing nucleic acid released by necrotic or late apoptotic cells and lupus IgG

Objective

To investigate the release of interferon‐α (IFNα)–inducing material by necrotic or apoptotic cells, its properties, and the necessity of autoantibodies from systemic lupus erythematosus (SLE) patients for the interferogenic activity.

Methods

U937 monocytic leukemia cells or peripheral blood mononuclear cells (PBMCs) were rendered necrotic by freeze‐thawing or apoptotic by treatment with ultraviolet light. Cell culture supernatants from these cells and IgG from SLE patients (SLE IgG) were added to cultures of normal PBMCs or purified plasmacytoid dendritic cells (PDCs). The importance of nucleic acids for IFNα induction was investigated by RNase and DNase treatment. The IFNα levels were measured by immunoassay.

Results

Both necrotic and apoptotic U937 cells released material that, combined with SLE IgG, induced IFNα production in PDCs. The release from apoptotic cells occurred with a 16‐hour delay, in late apoptosis. Also, normal PBMCs released IFNα‐inducing material, but only during necrosis. The interferogenic activity of the necrotic material required the presence of RNA, while both RNA and DNA were important in the apoptotic material. In both cases, the presence of SLE IgG was necessary, and its activity correlated with the presence of antibodies to RNA‐binding proteins, but not anti‐DNA antibodies.

Conclusion

Necrotic and late apoptotic cells release material that, combined with SLE IgG, induces production of IFNα in PDCs. The IFNα inducers probably consist of immune complexes (ICs) containing RNA and possibly DNA as essential interferogenic components. The presence of such interferogenic ICs could explain the ongoing production of IFNα in SLE and could be of etiopathogenic importance.

     

Interferon‐α–inducible proteins are novel autoantigens in murine lupus

Objective

To investigate the spectrum of B cell autoimmunity in the recently described anti‐CD1–autoreactive T cell receptor (TCR)–transgenic murine lupus‐like (CD1 lupus‐like) model.

Methods

Lethally irradiated BALB/c/nu/nu mice were injected intravenously with donor BALB/c bone marrow and spleen cells expressing TCRα and TCRβ transgenes that recognize CD1d. Sera from adoptive host animals that developed lupus (i.e., CD1 lupus mice) were collected at serial time points and analyzed by Western blotting and immunoprecipitation, using protein extracts prepared from NIH3T3 mouse fibroblasts and EL‐4 lymphocytes, respectively. Sera obtained from older animals in several models of spontaneous lupus (NZB/NZW, MRL++, and MRL/lpr mice), unmanipulated BALB/c/nu/nu mice, and normal BALB/c mice were used as controls.

Results

Analyses demonstrated that the prominent targets of autoantibodies in the CD1 lupus‐like model are interferon‐α (IFNα)–inducible antigens. Biochemical and serologic characterizations identified one antigen as belonging to the interferon‐inducible 202 (Ifi202) subfamily of proteins within the Ifi200 family, and a second antigen as a member of the 70‐kd heat‐shock protein family. Autoantibodies directed against these antigens were rapidly produced at an early stage of disease. Anti‐p50 autoantibodies were present in sera from 7 (78%) of 9 CD1 lupus mice that developed severe kidney disease.

Conclusion

IFNα‐inducible proteins represent a novel class of autoantigens in murine lupus, and the findings suggest additional roles for IFNα in this disease. Since Ifi202 autoantigens are encoded by the murine non–major histocompatibility complex lupus‐susceptibility gene locus Ifi202, these data provide a link between recent advances in lupus genetics and the formation of autoantibodies.

     

Association of endogenous anti–interferon‐α autoantibodies with decreased interferon‐pathway and disease activity in patients with systemic lupus erythematosus

Objective

Numerous observations implicate interferon‐α (IFNα) in the pathophysiology of systemic lupus erythematosus (SLE); however, the potential impact of endogenous anti‐IFNα autoantibodies (AIAAs) on IFN‐pathway and disease activity is unclear. The aim of this study was to characterize IFN‐pathway activity and the serologic and clinical profiles of AIAA‐positive patients with SLE.

Methods

Sera obtained from patients with SLE (n = 49), patients with rheumatoid arthritis (n = 25), and healthy control subjects (n = 25) were examined for the presence of AIAAs, using a biosensor immunoassay. Serum type I IFN bioactivity and the ability of AIAA‐positive sera to neutralize IFNα activity were determined using U937 cells. Levels of IFN‐regulated gene expression in peripheral blood were determined by microarray, and serum levels of BAFF, IFN‐inducible chemokines, and other autoantibodies were measured using immunoassays.

Results

AIAAs were detected in 27% of the serum samples from patients with SLE, using a biosensor immunoassay. Unsupervised hierarchical clustering analysis identified 2 subgroups of patients, IFN^low and IFN^high, that differed in the levels of serum type I IFN bioactivity, IFN‐regulated gene expression, BAFF, anti–ribosomal P, and anti‐chromatin autoantibodies, and in AIAA status. The majority of AIAA‐positive patients had significantly lower levels of serum type I IFN bioactivity, reduced downstream IFN‐pathway activity, and lower disease activity compared with the IFN^high patients. AIAA‐positive sera were able to effectively neutralize type I IFN activity in vitro.

Conclusion

Patients with SLE commonly harbor AIAAs. AIAA‐positive patients have lower levels of serum type I IFN bioactivity and evidence for reduced downstream IFN‐pathway and disease activity. AIAAs may influence the clinical course in SLE by blunting the effects produced by IFNα.

     

The DNA mismatch repair enzyme PMS1 is a myositis‐specific autoantigen

Objective

The specificity of the autoantibody response in different autoimmune diseases makes autoantibodies useful for diagnostic purposes. It also focuses attention on tissue‐ and event‐specific circumstances that may select unique molecules for an autoimmune response in specific diseases. Defining additional phenotype‐specific autoantibodies may identify such circumstances. This study was undertaken to investigate the disease specificity of PMS1, an autoantigen previously identified in some sera from patients with myositis.

Methods

We used immunoprecipitation analysis to determine the frequency of autoantibodies to PMS1 in sera from patients with myositis, systemic lupus erythematosus, or scleroderma and from healthy controls. Additional antigens recognized by PMS1‐positive sera were further characterized in terms of their susceptibility to cleavage by apoptotic proteases.

Results

PMS1, a DNA mismatch repair enzyme, was identified as a myositis‐specific autoantigen. Autoantibodies to PMS1 were found in 4 of 53 patients with autoimmune myositis (7.5%), but in no sera from 94 patients with other systemic autoimmune diseases (P = 0.016). Additional mismatch repair enzymes (PMS2, MLH1) were targeted, apparently independently. Sera recognizing PMS1 also recognized several other proteins involved in DNA repair and remodeling, including poly(ADP‐ribose) polymerase, DNA‐dependent protein kinase, and Mi‐2. All of these autoantigens were efficiently cleaved by granzyme B, generating unique fragments not observed during other forms of cell death.

Conclusion

PMS1 autoantibodies are myositis specific. The striking correlation between an immune response to a group of granzyme B substrates (functioning in DNA repair and remodeling) and the myositis phenotype strongly implies that tissue‐ and event‐specific biochemical events play a role in selecting these molecules for an autoimmune response. Understanding the role of granzyme B cleavage in this response is an important priority.

     

Sera from anti–Jo‐1–positive patients with polymyositis and interstitial lung disease induce expression of intercellular adhesion molecule 1 in human lung endothelial cells

Objective

To investigate whether sera or purified IgG from patients with polymyositis (PM) and patients with dermatomyositis (DM), with or without interstitial lung disease (ILD), can activate endothelial cells (ECs).

Methods

Patients' sera were selected based on the presence or absence of anti–Jo‐1, anti‐SSA, or anti–U1 small nuclear RNP autoantibodies. The presence of autoantibodies was determined by line blot assays. Cultured human microvascular ECs derived from lung tissue (HMVEC‐L) were incubated with sera or purified IgG from 22 patients with PM, 7 patients with DM, and 10 healthy individuals as controls. Assessment of intercellular adhesion molecule 1 (ICAM‐1) expression was conducted by immunofluorescence (n = 22) and by cell‐based enzyme‐linked immunosorbent assay (ELISA) (n = 20). Serum levels of soluble ICAM‐1 (sICAM‐1) were determined by ELISA.

Results

Sera from PM patients with ILD who were positive for anti–Jo‐1 autoantibodies had a significantly stronger effect on the expression of ICAM‐1 by HMVEC‐L in comparison with sera from healthy controls and patients with other autoantibodies. Purified IgG did not induce ICAM‐1 expression. Higher serum levels of sICAM‐1 were found in patients with myositis compared with healthy controls.

Conclusion

EC activation with ICAM‐1 expression could contribute to the multiorgan involvement, including the development of myositis and ILD, in patients carrying anti–Jo‐1 autoantibodies. The EC‐activating factors are not the autoantibodies themselves, but might be systemic factors associated with these autoantibodies.

     

Identity of the RNase MRP– and RNase P–associated Th/To autoantigen

Objective

To characterize the molecular identity of the Th/To autoantigen, which is targeted by autoantibodies in scleroderma and which is associated with the human RNase MRP and RNase P ribonucleoprotein complexes.

Methods

Proteins immunoprecipitated by anti‐Th/To+ patient antisera from biotinylated total HeLa cell extracts were analyzed by immunoblotting. The association of autoantigenic proteins with the RNase MRP complex was analyzed by reconstitution experiments and ultraviolet crosslinking. The reactivity of patient sera with all known RNase MRP/RNase P proteins was analyzed by immunoprecipitation of the individual recombinant proteins.

Results

The previously defined Th40 autoantigen appeared to be identical to the Rpp38 protein. Paradoxically, Rpp38 did not bind to the P3 domain of the RNase MRP RNA, as suggested by previously published data for Th40, and only half of the anti‐Th/To+ sera contained anti‐Rpp38 reactivity. Two other RNase MRP/RNase P subunits, Rpp20 and Rpp25, were found to interact with the P3 domain. The previously reported 40‐kd species associated with this domain appeared to consist of Rpp20 and/or Rpp25 associated with a nuclease‐resistant RNA fragment. Finally, we demonstrated that almost all tested anti‐Th/To+ patient sera contained autoantibodies to Rpp25 and hPop1, indicating that these proteins harbor the most frequently targeted Th/To determinants.

Conclusion

Our data unequivocally define the identity of the Th/To autoantigen and demonstrate that Th/To autoepitopes are found on several protein subunits of RNase MRP/RNase P.

     

RhoA‐mediated,tumor necrosis factor α–induced activation of NF‐κB in rheumatoid synoviocytes: Inhibitory effect of simvastatin

Objective

Increasing evidence indicates that RhoA may play a central role in the inflammatory response. This study was conducted to examine the role of RhoA in mediating the activation of NF‐κB in tumor necrosis factor α (TNFα)–stimulated rheumatoid synoviocytes, and to evaluate the modulatory effects of statins on the TNFα‐induced activation of RhoA and NF‐κB and the secretion of proinflammatory cytokines by rheumatoid synoviocytes.

Methods

Rheumatoid synoviocytes obtained from patients with active rheumatoid arthritis were stimulated with TNFα and incubated with simvastatin (SMV) (1 μM). RhoA activity was assessed by a pull‐down assay. NF‐κB DNA binding activity and nuclear translocation of NF‐κB were measured by a sensitive multiwell colorimetric assay and confocal fluorescence microscopy, respectively.

Results

TNFα stimulation elicited a robust increase in RhoA activity in a dose‐dependent manner, and SMV mitigated this increase. TNFα also hastened NF‐κB nuclear translocation of subunit p65 and increased DNA binding activity, luciferase reporter gene expression, degradation of IκB, and secretion of interleukin‐1β (IL‐1β) and IL‐6. SMV prevented the increase in NF‐κB activation and rise in IL‐1β and IL‐6 levels induced by TNFα, whereas mevalonate and geranylgeranyl pyrophosphate reversed the inhibitory effects of SMV on activation of NF‐κB and RhoA. Furthermore, cotransfection with a dominant‐negative mutant of RhoA demonstrated that the TNFα‐induced signaling pathway involved sequential activation of RhoA, leading to NF‐κB activation and, ultimately, to secretion of cytokines.

Conclusion

This study identifies RhoA as the key regulator of TNFα‐induced NF‐κB activation, which ultimately results in the secretion of proinflammatory cytokines in rheumatoid synoviocytes. The findings provide a new rationale for the antiinflammatory effects of statins in inflammatory arthritis.

     

Functional assay of type I interferon in systemic lupus erythematosus plasma and association with anti–RNA binding protein autoantibodies

Objective

Peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) have increased expression of genes typically induced by type I interferon (IFN). However, it has been difficult to identify and quantify the factors responsible for activation of the IFN pathway in SLE. To characterize these mediators, we developed an assay that measures the functional effects of plasma or serum components on the gene expression of cultured target cells.

Methods

WISH epithelial cell line cells were cultured with medium, with recombinant IFNα, IFNβ, or IFNγ, or with 50% plasma from SLE patients (n = 73), rheumatoid arthritis (RA) patients (n = 19), or healthy donors (n = 30). Real‐time quantitative polymerase chain reaction was used to determine WISH cell expression of IFN target genes, including PRKR, IFIT1, IFI44, MX1, and C1orf29 (preferentially induced by IFNα) and CXCL9 (Mig) (preferentially induced by IFNγ).

Results

IFNα‐regulated genes were induced by SLE plasma samples, but not by most of the RA or healthy control plasma samples. The activity in SLE plasma was inhibited >90% by anti‐IFNα antibody, but not by anti‐IFNβ or anti‐IFNγ antibodies. The expression of each IFNα target gene induced by SLE plasma correlated with the expression of that gene studied ex vivo in PBMCs from the same patients and with the titer of anti–RNA binding protein (anti‐RBP)–specific autoantibodies. Plasma activity paralleled PBMC expression of IFNα‐inducible genes over time.

Conclusion

IFNα in SLE plasma is a major stimulus of IFN target gene expression and is related to expression of those genes in PBMCs from SLE patients and to the titer of anti‐RBP autoantibodies. These data provide additional support for the view that IFNα mediates immune system activation and dysregulation in SLE.

     

Tumor necrosis factor α–mediated cleavage and inactivation of SirT1 in human osteoarthritic chondrocytes

Objective

The protein deacetylase SirT1 positively regulates cartilage‐specific gene expression, while the proinflammatory cytokine tumor necrosis factor α (TNFα) negatively regulates these same genes. This study was undertaken to test the hypothesis that SirT1 is adversely affected by TNFα, resulting in altered gene expression.

Methods

Cartilage‐specific gene expression, SirT1 activity, and results of chromatin immunoprecipitation analysis at the α2(I) collagen enhancer site were determined in RNA, protein extracts, and nuclei of human osteoarthritic chondrocytes left untreated or treated with TNFα. Protein extracts from human chondrocytes transfected with epitope‐tagged SirT1 that had been left untreated or had been treated with TNFα were analyzed by immunoblotting with SirT1 and epitope‐specific antibodies. The 75‐kd SirT1‐reactive protein present in TNFα‐treated extracts was identified by mass spectroscopy, and its amino‐terminal cleavage site was identified via Edman sequencing. SirT1 activity was assayed following an in vitro cathepsin B cleavage reaction. Cathepsin B small interfering RNA (siRNA) was transfected into chondrocytes left untreated or treated with TNFα.

Results

TNFα‐treated chondrocytes had impaired SirT1 enzymatic activity and displayed 2 forms of the enzyme: a full‐length 110‐kd protein and a smaller 75‐kd fragment. The 75‐kd SirT1 fragment was found to lack the carboxy‐terminus. Cathepsin B was identified as the TNFα‐responsive protease that cleaves SirT1 at residue 533. Reducing cathepsin B levels via siRNA following TNFα exposure blocked the generation of the 75‐kd SirT1 fragment.

Conclusion

These data indicate that TNFα, a cytokine that mediates joint inflammation in arthritis, induces cathepsin B–mediated cleavage of SirT1, resulting in reduced SirT1 activity. This reduced SirT1 activity correlates with the reduced cartilage‐specific gene expression evident in these TNFα‐treated cells.

     

Anti‐La/SSB antibodies transported across the placenta bind apoptotic cells in fetal organs targeted in neonatal lupus

Objective

To determine whether La and/or Ro epitopes on apoptotic cells in fetal organs that are targeted in neonatal lupus syndrome (NLS) are accessible for binding by autoantibodies in vivo, we traced the fate of transplacental autoantibodies in a murine passive transfer model.

Methods

Pregnant mice at day 15 of gestation (E15) were injected intraperitoneally with human anti‐Ro/La–positive sera or control sera, and transplacental transfer of human autoantibodies was tested by enzyme‐linked immunosorbent assay with recombinant antigens. Multiple cryostat sections at the level of the heart of E17 fetuses were visualized simultaneously for human IgG binding and apoptosis (TUNEL) under confocal microscopy. Serial paraffin sections of E17 and E19 fetuses were examined for histologic evidence of inflammation.

Results

Human IgG anti–52‐kd Ro, anti–60‐kd Ro, and anti‐La autoantibodies were transported efficiently into the fetal circulation. Human IgG–apoptotic cell complexes were detected in the heart (atrial trabeculae and atrioventricular node), skin, liver, and newly forming bone of fetuses from mothers injected with anti‐Ro/La sera but not control sera. The IgG binding was fetal‐specific and organ‐specific; transplacental autoantibodies did not bind to apoptotic cells in the fetal thymus, lung, brain, or gut. The complexes were not associated with an inflammatory reaction. Injection of mothers with affinity‐purified anti‐La autoantibodies (but not anti‐Ro/La Ig depleted of anti‐La) revealed an identical location of IgG binding to apoptotic cells in the fetuses.

Conclusion

This is the first study to demonstrate that transplacental anti‐La autoantibodies bind specifically to apoptotic cells in selected fetal organs in vivo, similar to the organ involvement in NLS. We hypothesize that additional factors are required to promote proinflammatory clearance of IgG–apoptotic cell complexes and subsequent tissue damage.

     

CYP7B expression and activity in fibroblast‐like synoviocytes from patients with rheumatoid arthritis: Regulation by proinflammatory cytokines

Objective

The cytochrome P450 enzyme CYP7B catalyzes the conversion of dehydroepiandrosterone (DHEA) into 7α‐hydroxy‐DHEA (7α‐OH‐DHEA). This metabolite can stimulate the immune response. We previously reported that the severity of murine collagen‐induced arthritis is correlated with CYP7B messenger RNA (mRNA) expression and activity in the arthritic joint. The purpose of this study was to investigate the presence of 7α‐OH‐DHEA in synovial samples and the cytokine regulation of CYP7B activity in fibroblast‐like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).

Methods

The presence of 7α‐OH‐DHEA was examined in synovial biopsy tissues, synovial fluid, and serum by radioimmunoassay. The effect of cytokines on CYP7B mRNA expression and CYP7B activity in FLS was examined by determining the formation of the CYP7B enzyme product 7α‐OH‐DHEA with the use of high‐performance liquid chromatography.

Results

The CYP7B enzyme product 7α‐OH‐DHEA was found in synovial biopsy tissues, synovial fluid, and serum from RA patients. The proinflammatory cytokines tumor necrosis factor α (TNFα), interleukin‐1α (IL‐1α), IL‐1β, and IL‐17 up‐regulated CYP7B activity in an FLS cell line 2–10‐fold. Enhanced CYP7B activity was correlated with an increase in CYP7B mRNA. The cytokine transforming growth factor β inhibited CYP7B activity. Moreover, CYP7B activity was detected in 10 of 13 unstimulated synovial fibroblast cell lines. Stimulation with TNFα increased CYP7B activity in all cell lines tested.

Conclusion

Exposure to the proinflammatory cytokines TNFα, IL‐1α, IL‐1β, and IL‐17 increases CYP7B activity in synovial tissue. Increased CYP7B activity leads to higher levels of the DHEA metabolite 7α‐OH‐DHEA in synovial fluid, which may contribute to the maintenance of the chronic inflammation observed in RA patients.

     

Estrogen receptor–related receptor α regulation by interleukin‐1β in prostaglandin E2– and cAMP‐dependent pathways in osteoarthritic chondrocytes

Objective

We reported previously that the orphan nuclear receptor, estrogen receptor–related receptor α (ERRα), is expressed in articular chondrocytes and is dysregulated in a mouse model of inflammatory arthritis. The aim of this study, therefore, was to determine whether ERRα is also dysregulated in patients with osteoarthritis (OA).

Methods

ERRα messenger RNA (mRNA) and protein were quantified in normal and OA cartilage samples and in OA chondrocytes in vitro, with and without short‐term treatment with a variety of OA‐associated factors and signaling pathway agonists and inhibitors.

Results

ERRα expression was lower in OA than in normal articular cartilage. Interleukin‐1β (IL‐1β) markedly up‐regulated ERRα expression in OA chondrocytes in vitro, and agonist or inhibitor treatment indicated that the up‐regulation was dependent on cyclooxygenase 2 (COX‐2; NS398), prostaglandin E₂, cAMP (8‐bromo‐cAMP), and protein kinase A (PKA; KT5720). Treatment with the ERRα inverse agonist XCT790 decreased the expression of SOX9 and the up‐regulation of ERRα by IL‐1β, suggesting autoregulation of ERRα in the IL‐1β pathway. Matrix metalloproteinase 13 (MMP‐13) expression was also decreased by treatment with XCT790 plus IL‐1β versus IL‐1β alone, and the down‐regulation of MMP‐13 mRNA and protein observed with XCT790 alone suggests that the up‐regulation of MMP‐13 by IL‐1β is ERRα‐dependent.

Conclusion

We report the first evidence that ERRα expression is regulated by IL‐1β in COX‐2–, cAMP‐, and PKA‐dependent pathways in OA chondrocytes. We confirmed that SOX9 is an ERRα target gene in human, as in rodent, chondrocytes and identified MMP‐13 as a potential new target gene, which suggests that ERRα may both respond to the healing signal and contribute to extracellular degradation in OA cartilage.