Minoru Satoh

AUTOANTIBODIES TO RNA POLYMERASE II ARE COMMON IN SYSTEMIC LUPUS ERYTHEMATOSUS AND OVERLAP SYNDROME : SPECIFIC RECOGNITION OF THE PHOSPHORYLATED (IIO) FORM BY A SUBSET OF HUMAN SERA

Autoantibodies to RNA polymerases (RNAP) I, II, and III are reported to be highly specific for the diagnosis of scleroderma (systemic sclerosis, SSc). In the present study, the specificity of autoantibodies to RNAP I and III for SSc was confirmed by immunoprecipitation of 35S-labeled proteins. However, we report here the previously unrecognized production of anti-RNAP II autoantibodies by 9-14% of patients with SLE and mixed connective tissue disease/overlap syndrome. 12 out of 32 anti-RNAP II positive sera (group 1) immunoprecipitated a diffuse 220-240-kD band identified as the largest subunit of RNAP II whereas the remaining 20 (group 2) immunoprecipitated preferentially the 240-kD phosphorylated (IIo) form of the large subunit. After pulse labeling, group 1 sera immunoprecipitated only the 220-kD (IIa) RNAP II subunit, whereas the diffuse IIa/IIo band plus the 145-kD second largest RNAP II subunit (IIc) were immunoprecipitated after several hours of cold chase, suggesting that these sera recognized primarily the largest subunit of RNAP II. Group 2 sera recognized the IIc subunit after pulse labeling, and immunoprecipitated the IIc and IIo, but not the IIa, subunits after cold chase. Although it has been suggested that autoantibodies to RNAP II are usually accompanied by anti-RNAP I/III in SSc, all but one of the anti-RNAP II positive sera from SLE or mixed connective tissue disease/overlap syndrome patients, as well as most of the SSc sera, were negative for anti-RNAP I/III. Moreover, in contrast to previous reports suggesting that anti-RNAP antibodies rarely coexist with other SSc subset marker antibodies, anti-RNAP II antibodies were often accompanied by anti-Ku, anti-nRNP, or anti-topoisomerase I autoantibodies in the present study. We conclude that autoantibodies to RNAP II are not a specific marker for SSc, whereas autoantibodies to RNAP I/III are associated primarily with SSc. In addition, we have identified two distinctive patterns of RNAP II antigen recognition by autoantibodies, one of them characterized by specific recognition of the transcriptionally active (phosphorylated) form of RNAP II. The clinical significance of these different patterns remains to be determined.

Autoantibodies to DEK oncoprotein in human inflammatory disease.

OBJECTIVEnTo evaluate the specificity of anti-DEK antibodies for juvenile rheumatoid arthritis (JRA).nnnMETHODSnAnti-DEK autoantibodies were measured by enzyme-linked immunosorbent assay (ELISA) using affinity-purified his6-DEK fusion protein. Sera from 639 subjects (417 patients with systemic autoimmune disease, 13 with sarcoidosis, 44 with pulmonary tuberculosis, 125 with uveitis, and 6 with scleritis, and 34 healthy control subjects) were screened. Reactivity was verified by immunoblotting and immunoprecipitation studies using baculovirus-expressed human DEK.nnnRESULTSnAnti-DEK activity was found at the following frequencies: JRA 39.4% (n = 71), systemic lupus erythematosus (SLE) 25.1% (n = 216), sarcoidosis 46.2% (n = 13), rheumatoid arthritis 15.5% (n = 71), systemic sclerosis 36.0% (n = 22), polymyositis 6.2% (n = 16), and adult Stills disease 0% (n = 21). Autoantibodies also were detected in 9.1% of tuberculosis sera (n = 44), but were undetectable in sera from the 34 healthy controls. Western blot and immunoprecipitation assay results correlated well with the ELISA findings. In general, levels of anti-DEK autoantibodies were higher in SLE than in other patient subsets, including JRA.nnnCONCLUSIONnAnti-DEK autoantibodies are less specific for JRA than previously believed. They are produced in association with a variety of inflammatory conditions, many of which are associated with granuloma formation and/or predominant Thl cytokine production. Anti-DEK antibodies may be a marker for a subset of autoimmunity associated with interferon-gamma production rather than a particular disease subset.

Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens

The Ku autoantigen is a DNA binding factor consisting of 70 and ∼80 kDa proteins (p70 and p80, respectively) which form a heterodimer. The p70/p80 dimer appears to be crucial for the function of a 350 kDa DNA-dependent protein kinase (DNA-PK) that phosphorylates certain transcription factorsin vitro. Previous studies have suggested that Ku is abundant in primate cells, but undetectable in most non-primate cells. However, it is unclear if this reflects low abundance of Ku (and possibly DNA-PK activity) in non-primate cells, a lack of antibodies crossreactive with non-primate Ku proteins, or both. Ku was first identified with human autoimmune sera, but the suitability of these sera for studying the distribution, abundance and function of Ku is limited by the polyclonal immune response to Ku and the presence of contaminating autoantibodies in most patients sera. In the present studies, we determined the specificities of murine anti-Ku monoclonal antibodies (mAbs) using cellular Ku as well as recombinant human and murine Ku antigens. Immunofluorescence studies confirmed previous observations that Ku is undetectable in most nonprimate cells. However, small amounts of Ku could be detected in MOPC-315, but not L-929, cells by immunoprecipitating with mAb 162. In addition, autoantibodies to Ku were identified in the sera of ∼1/3 of MRL/lpr mice. The murine autoantibodies also immunoprecipitated a small amount of Ku (comparable to that seen with 162) from MOPC-315, but not L-929, cell lysates. Characterization of the mAb specificities by immunoblot analysis with Ku fusion proteins revealed that mAbs 111, S10B1, and N9C1 bound to distinct epitopes of human p80 (amino acids 610–705, 8–221, and 1–374, respectively). All three mAbs were unreactive with murine p80. MAbs N3H10 and S5C11 bound immediately adjacent to the DNA binding site of p70 (amino acids 506–541). Only N3H10 displayed comparable reactivity with human and murine p70 on immunoblots, but it immunoprecipitated murine Ku poorly. S5C11 crossreacted more weakly with murine p70 on immunoblots, whereas 162 was completely unreactive with human or murine Ku on immunoblots, despite immunoprecipitating Ku efficiently. Studies with mAbs N3H10 and 162 suggest that the level of Ku is considerably lower in nonprimate cells than cells of primate origin, and that L-929 cells express little or no Ku protein. These mAbs constitute a panel of immunological reagents reactive with defined regions of the Ku autoantigen which should be useful for examining the assembly and function of Ku.

Pristane induces high titers of anti-Su and anti-nRNP/Sm autoantibodies in BALB/c mice : quantitation by antigen capture ELISAs based on monospecific human autoimmune sera

Autoantibodies to Su and anti-nRNP/Sm are common in human and murine systemic lupus erythematosus (SLE), and are also produced by BALB/c mice with SLE-like autoimmunity induced by pristane. Antigen capture ELISAs employing monospecific human autoimmune IgG were developed to quantitate the production of anti-Su and anti-nRNP/Sm autoantibodies in 77 sera from BALB/c mice with pristane-induced autoimmunity. The sensitivity and specificity of the anti-Su antigen capture ELISA were 100% compared with immunoprecipitation of 35S-labeled cellular proteins. All 16 immunoprecipitation positive sera were positive in the anti-nRNP/Sm antigen capture ELISA (100% sensitivity), whereas 55/61 immunoprecipitation negative sera were negative by ELISA (90% specificity). The 6/61 immunoprecipitation negative sera that were ELISA positive were probably true positives because subsequent sera obtained from the same mice were positive by both techniques. Thus, the antigen capture ELISA may be somewhat more sensitive than immunoprecipitation. The titers of anti-Su and anti-nRNP/Sm positive antibodies in the sera were as high as 1:25,000-1:250,000 by ELISA, suggesting that autoantibodies may be produced in pristane-primed BALB/c mice at levels comparable to those seen in spontaneous autoimmune disease. We conclude that antigen capture ELISAs based on human autoimmune sera were highly sensitive and specific for detecting murine anti-Su and anti-nRNP/Sm antibodies. This technique will be useful for quantitating antibodies in murine autoimmune disease models, since antigen capture ELISA avoids the use of denatured or recombinant antigens, permitting antibodies recognizing tertiary and quaternary structures to be detected.

Autoantibodies that stabilize the molecular interaction of Ku antigen with DNA-dependent protein kinase catalytic subunit

DNA‐dependent protein kinase (DNA‐PK) consists of a DNA binding subunit (Ku autoantigen), and a catalytic subunit (DNA‐PKcs). In the present study, human autoantibodies that recognize novel antigenic determinants of DNA‐PK were identified. One type of autoantibody stabilized the interaction of DNA‐PKcs with Ku and recognized the DNA‐PKcs–Ku complex, but not biochemically purified DNA‐PKcs. Another type recognized purified DNA‐PKcs. Autoantibodies to Ku (p70/p80 heterodimer), ‘stabilizing’ antibodies, and antibodies to DNA‐PKcs comprise a linked autoantibody set, since antibodies recognizing purified DNA‐PKcs were strongly associated with stabilizing antibodies, whereas stabilizing antibodies were strongly associated with anti‐Ku. This hierarchical pattern of autoantibodies specific for components of DNA‐PK (anti‐Ku>stabilizing antibodies>anti‐DNA‐PKcs) may have implications for the pathogenesis of autoimmunity to DNA‐PK and other chromatin particles. The data raise the possibility that altered antigen processing and/or stabilization of the DNA‐PKcs–Ku complex due to autoantibody binding could play a role in spreading autoimmunity from Ku to the weakly associated antigen DNA‐PKcs.

Similar DNA binding properties of free p70 (Ku) subunit and p70/p80 heterodimer

The Ku antigen consists of 70 and 80 kDa protein subunits (p70 and p80, respectively) that form the DNA binding component of a DNA‐dependent protein kinase (DNA‐PK). It is controversial whether the interaction of Ku with DNA is mediated by p70 alone or requires formation of p70/p80 dimers. In the present studies, the DNA binding properties of p70/p80 heterodimers and full‐length human p70 expressed in the absence of p80 were investigated. The binding of free p70 and p70/p80 heterodimers to DNA showed similar sensitivity to high ionic strength buffers. Competitive DNA binding studies revealed that free p70, like the p70/p80 heterodimer, bound preferentially to linear double stranded DNA fragments, whereas tRNA and closed circular DNA molecules competed poorly with the radiolabeled linear DNA for binding to Ku. These studies suggest that free p70 and p70/p80 heterodimers have similar DNA binding properties, and that the interaction of Ku with DNA may depend primarily on the p70 subunit, possibly with implications for the assembly and function of DNA‐PK.

15 – ORIGINS OF ANTINUCLEAR ANTIBODIES

Publisher Summary The production of antinuclear antibodies (ANAs) is one of the defining features of systemic lupus erythematosus (SLE). This chapter reviews what has been learned about antinuclear antibodies and their molecular targets and pathogenesis. Interferons, cytokines with antiviral and antiproliferative effects, as well as important effects on the activation of immune effector cells, are likely to be involved in the pathogenesis of SLE. They are classified into type I and type II IFNs based on sequence homology, receptor usage, and the cellular origin. There is strong evidence that T cells are involved in autoantibody production, including indirect evidence from characteristics of the autoantibody response, the inability of tetramethylpentadecane (TMPD) to induce autoantibodies in T-cell-deficient mice, the likely involvement of TH17 and TFH cells in the induction of lupus autoantibodies, and the diminished autoantibody production following CTLA4-Ig treatment. However, there also is evidence that autoantibodies can arise independently of T cells via extrafollicular activation of autoreactive B cells. Most studies suggest that B-1 cells produce polyreactive antibodies, exhibit only limited somatic mutation, develop independently of T cells, and are prone to make low-affinity autoantibodies against repetitive epitopes such as pneumococcal polysaccharide (TI-2 antigens). In contrast, conventional (B-2) B cells require cognate T cell help and produce high-affinity, somatically mutated antibodies. Further studies are needed to define to what degree autoantibody production in SLE patients results from cognate T–B interactions and post-germinal center memory/plasma cells and vs. extrafollicular, T-cell-independent (TLR-mediated) responses.

Human autoantibodies stabilize the quaternary structure of Ku antigen

OBJECTIVEnTo examine humoral immune responses to the native Ku antigen and to evaluate the role of autoantibodies in stabilizing intermolecular contacts between the p70 and p80 Ku subunits.nnnMETHODSnRecombinant free human p70 and p80 Ku subunits and p70/p80 heterodimers were expressed in Sf9 (insect) cells using baculovirus vectors. Affinity-purified recombinant human p70, p80, and p70/p80 dimer were studied by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation to evaluate autoantibody specificities in sera from 58 patients with systemic autoimmune disease.nnnRESULTSnAnti-Ku antibodies were detected by ELISA or immunoprecipitation using K562 cell Ku antigen. All of the sera were reactive with the native recombinant p70, p80, or p70/p80 antigens: 47% were anti-p70+,anti-p80+ and 32% were anti-p70-,anti-p80+, but only 3% were anti-p70+,anti-p80-. Unexpectedly, 18% of the sera recognized the p70/p80 dimer but did not recognize native p70 or p80 alone. A subset of sera containing autoantibodies that prevent the dissociation of p70 from p80 by high salt and detergent treatment was identified; monoclonal antibody (MAb) 162, a murine anti-Ku MAb, displays the same property. Autoantibodies that stabilize the p70-p80 interaction were found most frequently in sera containing both anti-p70 and anti-p80 antibodies.nnnCONCLUSIONnAutoantibodies to the native p80 subunit of Ku are more common than are anti-p70 antibodies. When anti-p70 antibodies were detected, they generally were found together with anti-p80. A novel type of autoantibody capable of stabilizing the p70/p80 heterodimer was identified in human sera for the first time. These stabilizing autoantibodies are found in sera containing both anti-p70 and anti-p80 antibodies, and also are produced by mice immunized with human Ku antigen. Autoimmunity to Ku may be initiated with an immune response to p80, followed by spreading to p70. We hypothesize that stabilizing antibodies could facilitate the spreading of autoimmunity from one subunit of Ku to another by altering the processing of p70 or p80 by antigen-presenting cells.

Correlation of antisynthetase antibody levels with disease course in a patient with interstitial lung disease and elevated muscle enzymes.

Antiglycyl tRNA synthetase is an unusual autoantibody specificity associated with polymyositis and dermatomyositis complicated by interstitial lung disease. We report here autoantibodies to glycyl tRNA synthetase in a patient with systemic lupus erythematosus and interstitial lung disease. During the course of her disease, the patient developed elevated muscle enzymes and worsening pulmonary function. A quantitative immunoprecipitation technique was developed to evaluate the relationship between autoantibody production and clinical manifestations in this patient. Increasing serum antiglycyl tRNA synthetase antibody levels correlated with the severity of the patients interstitial lung disease and with the level of creatine phosphokinase.These results suggest that certain autoantibodies, such as antiglycyl tRNA synthetase, might reflect an underlying pathologic process (in this case, myositis and interstitial lung disease) irrespective of disease diagnosis, and that quantitative immunoprecipitation may be a useful technique for investigating the relationship between specific autoantibody production and organ involvement in systemic autoimmune disease, Quantitation of these autoantibodies may be useful clinically in monitoring disease activity and/or response to therapy.

FEATURES OF AUTOANTIGENS

The major cellular antigens recognized by autoantibodies in SLE and other systemic autoimmune diseases have been identified and characterized over the past 25 years. The pioneering studies of Eng Tan demonstrate the importance of autoantibodies as diagnostic markers. However, why certain autoantibodies, such as anti-Sm, are pathognomonic of SLE, while others are markers of othe autoimmune disease subsets, remains unanswered. This central question continues to drive much current research into the pathogenesis of SLE. Features of the autoantigens recognized by autoantibodies may provide important clues to the causes of lupus. Most autoantigens in systemic autoimmunity are multicomponent nucleoprotein complexes. These particles are encountered by the immune system as units, resulting in the tandem production of autoantibodies recognizing several components of the same complex. However, the intermolecular-intrastructural spreading of autoimmunity is regulated by mechanisms that at present are defined poorly. Also unexplained is the observation that the antigenic determinants recognized by autoantibodies are restricted and frequently correspond to active sites or functional domains. Analysis of experimental models of autoimmunity suggests that altering the structure of autoantigens, due to abnormal protein-protein interactions, hapten binding, altered degradation, or other mechanisms, could help to explain both the restricted patterns of autoantibody spreading and the selective targeting of antigenic sites. This may be a worthwhile area for further investigation of the pathogenesis of systemic autoimmune diseases.