Use of monoclonal antibodies for the characterization of novel DNA-binding proteins recognized by human autoimmune sera

Autoantibodies to a DNA-binding heterodimer consisting of 70,000 and 80,000 dalton subunits were identified in 30-50% of human autoimmune sera from patients with systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), and scleroderma. Three murine monoclonal antibodies (mAb) against the heterodimer were produced in BALB/c mice by immunizing with isolated human B cell nuclei. By immunofluorescence, the mAb and autoimmune sera demonstrated both speckled nucleoplasmic staining and diffuse nucleolar staining in all human cell types examined. The nucleoplasmic staining was sensitive to DNase but not RNase pretreatment, while the nucleolar staining was sensitive to RNase but not DNase pretreatment. Biochemical characterization of the 70,000 and 80,000 dalton proteins using the mAb indicated that two forms of the antigen, with different mobilities on sucrose gradients, are present in human B cells. A 10 S form consists of the physically associated 70,000 and 80,000 dalton proteins, while a larger, 10-20 S form probably represents the same two proteins bound to DNA. Binding of the proteins to nucleolar RNA could not be confirmed in biochemical studies. These studies indicate that non-histone, DNA-binding proteins may be more frequently recognized by autoantibodies in SLE, MCTD, and scleroderma than has been previously recognized. Along with previous studies on RNA-binding proteins such as Sm, RNP, Ro, and La, the present findings suggest that nucleic acid-binding proteins, as a class, may be particularly frequent targets of autoimmunity in SLE and related disorders.     

Anti-RNA polymerase I antibodies in sera of MRL lpr/lpr and MRL +/+ autoimmune mice. Correlation of antibody production with delayed onset of lupus-like disease in MRL +/+ mice

Sera from individual MRL/lpr and MRL/++ mice, which develop an autoimmune disease similar to human systemic lupus erythematosus (SLE), were screened over a period of approximately 30 wk for the presence of anti-RNA polymerase I and anti-ssDNA antibodies. Even though onset of the disease is delayed in MRL/++ as compared to MRL/lpr mice, anti-ssDNA antibodies were present in comparable concentrations in the sera of all mice by the age of 6 wk. As observed in sera of human SLE patients, anti-RNA polymerase I antibodies were detected in the sera of all MRL mice. However, unlike the anti-ssDNA antibodies, anti-RNA polymerase I antibodies were detected much later in MRL/++ mice (mean age, 22.8 wk) as compared to MRL/lpr mice (mean age, 9.6 wk). The presence of anti-RNA polymerase I antibodies in sera of MRL mice was thus a much better indicator of disease status than the presence of anti-ssDNA antibodies. The appearance and increase in anti-RNA polymerase I antibodies in the sera of MRL/++ mice correlated (R2 = 0.964) with a precipitous decrease in anti-ssDNA antibodies, starting at about 20 wk of age. These results suggest a possible relationship between the RNA polymerase I and DNA autoimmune reactions.     

Detection of autoantibodies to ss-a/ro by indirect immunofluorescence using a transfected and overexpressed human 60 kd ro autoantigen in hep-2 cells

The objective of the study was to determine the sensitivity and specificity of an indirect immunofluorescence (IIF) assay using transfected HEp-2 cells to detect anti-SS-A/Ro autoantibodies in human sera. Seventy-three sera having SS-A/Ro autoantibodies as determined by double immunodiffusion (ID) and immunoblotting (IB) were tested by IIF on a HEp-2 cell substrate that had been transfected with a full-length cDNA encoding a human 60 kD SS-A/Ro autoantigen. Controls included 30 normal human sera and 50 sera with a variety of other antinuclear antibodies. Prototype human and rabbit sera directed against the 60 kD SS-A/Ro antigen produced intense speckled nuclear and nucleolar staining of transfected cells. Sixty-nine of 73 (95%) SS-A/Ro positive sera also produced this characteristic staining pattern. The endpoint autoantibody titers on transfected cells was fivefold greater than on untransfected cells. The 30 normal human sera and the 50 sera with other antinuclear antibodies did not produce this characteristic staining. Six of 32 (19%) unselected sera that were sent for autoantibody testing had reactivity with transfectants by IIF. Four of the six sera were confirmed to have anti-SS-A/Ro antibodies by ID and 5/6 by IB. By contrast, only three of these sera were scored as having a staining pattern compatible with SS-A/Ro antibodies by IIF on standard HEp-2 substrates. We conclude that SS-A/Ro autoantibodies can be detected by an IIF assay using a HEp-2 cell substrate transfected with a SS-A/Ro cDNA. This new substrate detects SS-A/Ro antibodies that were not identified on standard HEp-2 substrates and by other immunoassays.©1995 wiley-Liss, inc.     

Molecular characterization of an autoantigen of PM-Scl in the polymyositis/scleroderma overlap syndrome: a unique and complete human cDNA encoding an apparent 75-kD acidic protein of the nucleolar complex

About 50% of patients with the polymyositis/scleroderma (PM-Scl) overlap syndrome are reported to have autoantibodies to a nuclear/nucleolar particle termed PM-Scl. The particle is composed of several polypeptides of which two have been identified as autoantigens. In this report, human cDNA clone coding for the entire 75-kD autoantigen of the PM-Scl particle (PM-Scl 75) was isolated from a MOLT-4 lambda gt-11 library. The deduced amino acid sequence of the cDNA clone represented a protein of 355 amino acids and 39.2 kD; the in vitro translation product of this cDNA migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at approximately 70 kD. The aberrant migration of the polypeptide in SDS-PAGE was shown to be related to the COOH half that was rich in acidic residues. Authenticity of the cDNA coding for PM-Scl 75 was shown by immunoreactivity of PM-Scl sera with in vitro translation products and recombinant fusion proteins encoded by the cDNA. In addition, rabbit antibodies raised to recombinant fusion protein reacted in immunofluorescence, immunoblotting, and immunoprecipitation with the characteristic features displayed by human anti-PM-Scl sera.     

Inhibition of antibodies binding polyinosinic polycytidylic acid in human and mouse lupus sera by viral and synthetic ribonucleic acids

The specificities of anti-RNA antibodies of diverse origin were studied by inhibition of the binding of radioative polyinosinic polycytidylic acid. The antibodies were from human patients with systemic lupus erythematosus (SLE), older NZB/NZW F(1) mice who have SLE, and young NZB/NZW F(1) mice immunized with either synthetic or viral double-stranded (ds) RNA. The inhibitors were two viral ds and two synthetic ds RNAs, ribosomal RNA and transfer RNA. The human sera were more heterogeneous than the mouse lupus sera, and had greatest specificity for reovirus RNA. The mouse lupus sera were more homogeneous and, in general, were inhibited efficiently by all four ds RNAs. Sera from mice immunized with synthetic RNA reacted poorly with viral RNA, whereas sera from mice immunized with viral RNA reacted with all four ds RNAs and resembled the lupus sera. These results suggest a role for viruses in the induction of anti-RNA antibodies, and are compatible with the concept that virus infection as well as excessive antibody responses are involved in the pathogenesis of SLE.     

Scl-86, a marker antigen for diffuse scleroderma.

More than 300 sera from patients with a connective tissue disease were analyzed with the immunoblotting technique. The presence of autoantibodies against an 86,000-mol wt marker antigen for diffuse scleroderma (Scl-86) was found in 14 out of 33 patients with scleroderma. The presence of anti-Scl-86 antibodies seemed to correlate with the diagnosis of diffuse scleroderma since they were found in 13 out of 22 diffuse scleroderma patients and in only one out of 11 patients with limited scleroderma. All scleroderma sera (33 patients' sera and 13 reference sera) were also tested for the presence of anti-Scl-70 antibodies. It was found that all anti-Scl-70 positive sera (n = 25) contained anti-Scl-86 antibody as well, suggesting a relationship between these two antigens. However, the Scl-86 antigen was shown to be an extremely insoluble nonchromosomal protein, resistant to boiling in sodium dodecyl sulfate. This contrasts with the Scl-70 antigen, which has been described as a thermolabile, soluble antigen present in the chromatin fraction. Together, our results are consistent with the idea that Scl-70 is a degradation product of Scl-86. The Scl-86 antigen is present in freshly prepared rabbit thymus, spleen, and liver nuclei as well as in nuclei from various cultured cell lines, but is not detectable in extractable nuclear antigen from rabbit thymus. In a limited retrospective study, the anti-Scl-86 antibodies were found in two sera from patients with Raynaud's phenomenon before the development of diffuse scleroderma. Therefore, it is possible that screening of patients' serum for this antibody might predict the development of diffuse scleroderma.     

Antimyenteric neuronal antibodies in scleroderma.

The pathogenesis of gastrointestinal (GI) dysmotility in scleroderma is incompletely understood, although previous studies have proposed a neuropathic mechanism. We studied patients with scleroderma as compared with other connective tissue disease patients and normal controls for the presence of circulating antibodies to myenteric neurons. Serial dilutions of sera were overlaid on rat intestine, double-labeled with antineurofilament antibody as a myenteric plexus marker, and imaged using indirect immunofluorescence techniques. High titer sera (> or = 1:50) from 19 out of 41 scleroderma patients stained myenteric neurons, whereas none of 22 normals or 5 patients with idiopathic GI dysmotility were positive. Although 6 out of 20 SLE and 6 out of 10 mixed connective tissue disease patients' sera stained myenteric plexus neurons, when positive sera were absorbed with calf thymus extract to remove antinuclear antibody, 15 scleroderma sera, 0 SLE, and 2 mixed connective tissue disease patients retained positive staining of myenteric neurons. Western blotting using actin and neuronal intermediate filament preparations failed to show immunoreactivity with scleroderma sera containing antimyenteric neuronal antibodies. Paraneoplastic sera associated with GI dysmotility stained myenteric neurons in a different pattern than seen with scleroderma sera. A positive correlation between the presence of Raynaud's phenomenon and antimyenteric neuronal antibodies was observed in scleroderma patients. Our results indicate that IgG antibodies reacting with myenteric neurons are present in many patients with scleroderma. Although the neuronal antigen has not yet been identified, the presence of myenteric neuronal antibodies in patients with GI dysmotility and scleroderma suggests a neuropathic process.     

The U2 small nuclear ribonucleoprotein particle as an autoantigen. Analysis with sera from patients with overlap syndromes.

We identified eight patients whose sera contained autoantibodies to the U2 small nuclear ribonucleoprotein (snRNP), an RNA protein particle involved in the splicing of newly transcribed messenger RNA. Each of these patients had an overlap syndrome that included features of either systemic lupus erythematosus (SLE), scleroderma, and/or polymyositis. We then used these sera to characterize the autoantigenic polypeptides of the U1 and U2 snRNP particles. In immunoblots, all sera contained antibodies to the B" polypeptide of the U2 snRNP. A subset of these sera that more effectively immunoprecipitated the native U2 particle contained an additional antibody system that recognized the A' polypeptide of this snRNP. Antibodies eluted from the B" protein bound the A polypeptide of the U1 snRNP and vice versa. Moreover, antibodies to the B" polypeptide were accompanied by antibodies to the 68K and C polypeptides of the U1 snRNP. Finally, the A' and B" polypeptides remained physically associated after the U2 particle was cleaved with RNase. Thus these sera contain multiple autoantibody systems that, at one level, target two physically associated antigenic polypeptides of the U2 particle and, at another, target two snRNP particles which are associated during the splicing of premessenger RNA. These linked autoantibody sets provide further evidence that intact macromolecular structures are targeted by the immune response in SLE and related diseases.     

Detection of anti-SSA antibodies by indirect immunofluorescence

BACKGROUND: HEp-2 cells that overexpress the human 60-kDa SSA antigen have been used to improve sensitivity and specificity for the detection of anti-SSA antibodies by indirect immunofluorescence. We describe a survey on the detection of anti-SSA antibodies using a commercial substrate that overexpresses SSA. METHODS: The evaluation was done on 18 371 consecutive samples submitted to the laboratory for detection of anti-nuclear antibodies, from which 188 anti-SSA antibody-containing and clinically documented samples were obtained. The presence of anti-SSA antibodies produced a distinct bright speckled pattern with nucleolar staining in 10-20% of interphase cells. The identity of all anti-SSA antibodies was confirmed by dot-blot analysis. RESULTS: Samples containing anti-SSA antibodies were separated into three main groups: group I, distinctive SSA pattern and other nuclear staining (50%); group II, only the distinctive SSA pattern (29%); group III, nuclear staining but without the distinctive SSA pattern (21%). Anti-SSA antibodies with concurrent SSB antibodies were associated with group I, whereas anti-SSA antibodies with concurrent U(1)-RNP antibodies were associated with group III. Group I included mainly patients with Sjogren syndrome and systemic lupus erythematosus (SLE), whereas group III included patients with mixed connective tissue disease and SLE. Diseases not classically associated with the presence of anti-SSA antibodies were found in group II in >50% of the cases. CONCLUSIONS: SSA-positive individuals were identified in a population selected on the basis of HEp-2000 positivity. Our study highlights diseases associated with anti-SSA antibodies and associations between the presence of the distinctive SSA pattern on HEp-2000 and some clinical conditions.     

Antinuclear antibodies: analysis of images and classification of immunofluorescence aspects on nuclei isolated from rat hepatocytes

Image analysis and Fourier transformation of the various nuclear immunofluorescence patterns observed while detecting antinuclear antibodies allow an objective and quantitative definition of the fluorescence. They also point out various IF types hidden by the main pattern, without having to dilute the test serum. They make obvious the difference between speckled and reticular patterns, and reveal the existence of intermediate states. The usual nuclear IF patterns (homogeneous, ring, nucleolar, reticulated, speckled and diffuse) may be grouped, according to their photo emission, into nuclear and subnuclear patterns. The first group includes homogeneous, annular and passive nucleolar IF. The second group is composed of speckled, reticulated, mixed, and active nucleolar IF. Alternatively, these aspects may be grouped into three types: homogeneous nuclear IF (homogeneous and ring), heterogeneous nuclear IF (speckled, reticulated and mixed) and nucleolar IF (active or passive). Diffusion can affect or not these aspects and does not apply to a special type or pattern. Image analysis and the study of the image spatial spectrum lead to automated recognition of the IF types, and later on, to the discrimination of antinuclear antibodies.     

Methods to detect antifibrillarin antibodies in patients with systemic sclerosis (SSc): a comparison

Autoantibodies against nucleolar antigens are common in systemic sclerosis (SSc). They include autoantibodies against fibrillarin (Fb), which are serological markers for SSc. Fb is associated with the evolutionally-conserved box C/D of small nucleolar RNAs (snoRNAs). We compared indirect immunofluorescence (IIF), Western blot (WB), and immunoprecipitation (IPP) of total small RNAs assays to determine which of these techniques is most specific for the detection of snoRNPs. We also examined the frequency and specificity of autoantibodies from SSc patients to snoRNAs, snRNAs, and scRNAs, and concluded that 1) IIF can not determine autoantibody specificity against Fb, 2) 36% of SSc sera were false-negative by WB, and 3) by IPP, anti-Fb autoantibodies from SSc patients can bind U3, U8, U13, U15, and U22 snoRNAs.     

Detection of antibodies in sera from patients with Opisthorchiasis

The indirect immunofluorescent antibody technique (IFA) was used for detection of antibodies in sera of patients with Opisthorchiasis. Antibodies to fluke worm and egg antigens were detected in 166 of 205 (81%) patients. The test showed that only the IgG class of antibodies reacting exclusively with integumental wall of the worm (AW) were positive in 46.8% (96/205), reacting only with the wall of intact eggs in 11 out of 205 (5.4%) and antibodies to both fluke and their egg antigens were present in 28.8% (59/205). In addition, 5.4% (11/205) of patients' sera were positive for autoantibodies producing a speckled antinuclear antibodies (ANA) pattern. The sera positive for only AW contained detectable autoantibodies to other cell antigens including: anti-smooth muscle antibodies of 9.4% (9/96), antimitochondrial antibodies of 3.1% (3/96), anti-liver/kidney microsomes of 1% (1/96) and anti-parietal cell antibodies of 1% (1/96). Autoantibodies were undetectable in sera from normal subjects. Among the ANA positive sera, 55% (6/11) exhibited antibodies against an extractable nuclear antigen (ENA) by a tanned red cell hemagglutination assay. This finding may suggest that the autoantibody response was due to the cross reaction between worm antigen and self antigen or it may be the result of polyclonal activation of B lymphocytes in these patients.     

Detection of anti-cerebral autoantibodies in schizophrenia and Alzheimer's disease

Autoantibodies to cerebrum were investigated in the sera from schizophrenia, Alzheimer's disease and healthy subjects by indirect immunofluorescence method. Four patterns of staining were observed: type A (neuronal cell), type B (blood vessel), type C (nucleus) and type D (glia and miscellaneous cell). Among rat, guinea pig and rabbit cerebrum, rabbit cerebrum proved best (low non-specific binding and high sensitivity) for the detection of circulating autoantibodies to cerebrum. With schizophrenia, 13 out of 46 cases (28.3%) were positive for autoantibodies to neuronal cells and 6/13 of those were not absorbed by liver acetone powder. With Alzheimer's disease, 25/63 (39.7%) were positive for autoantibodies to neuronal cells and 6/25 were not absorbed by liver acetone powder.     

Lupus antiribosomal P antisera contain antibodies to a small fragment of 28S rRNA located in the proposed ribosomal GTPase center

The ribosomal P proteins are necessary for GTPase activity during protein synthesis. In addition to antibodies to the P proteins, sera from lupus patients contain anti-rRNA activity. To determine whether lupus antiribosomal sera recognize the region of 28S rRNA recently proposed to form part of the ribosomal GTPase center, an rRNA fragment corresponding to nucleotides (nt) 1922-2020 was transcribed in vitro and tested for antigenicity. 18 of 24 (75%) lupus sera containing anti-P antibodies, but only 2 of 24 (8%) lupus sera without anti-P, immunoprecipitated this rRNA fragment (p less than 0.001). The binding was specific, since no significant differences were observed between anti-P positive and negative lupus sera in binding to the RNA fragment transcribed in the antisense orientation or to a control region of rRNA. The majority of sera tested protected a rRNA fragment of approximately 68 nucleotides. To evaluate the fine specificity of the anti-28S antibodies, deletions and site-directed mutations were made in the RNA fragment. The anti-28S antisera required nt 1944-1955 for recognition and were remarkably sensitive to destabilizing as well as nondestabilizing mutations in the stems of the RNA fragments. Detection of antiprotein and anti-RNA antibodies directed against a functionally related domain in the ribosome, together with the remarkable specificity of anti-28S antibodies, strongly suggests a direct role for this region of the ribosome in initiating and/or maintaining antiribosomal autoantibody production.     

Antibodies to granulocytes detected by an indirect immunofluorescence method not requiring chemical modification of cells

IgG antibodies to neutrophil polymorphonuclear cells (PMNs) were detected by a simple indirect immunofluorescence method that did not require chemical or enzymatic modification of the cells. Based on recent information about the human PMN Fc gamma-receptor, nonspecific binding of IgG to PMNs was virtually completely prevented by incubating cells at 37 degrees C with human serum diluted 1 to 3 in the presence of a high concentration of rabbit IgG. This also facilitated distinction of sera with weak anti-PMN activity from normal sera. On the other hand, nonspecific staining remained on a fraction (10-25%) of mononuclear leukocytes. Following first-time transfusion of 0.5 to 5 units of whole blood or red cell concentrate, 4 of 27 patients (15%) developed anti-leukocyte antibodies. Anti-leukocyte antibodies were detected in 51 percent of 47 patients with reported febrile transfusion reactions; the IgG antibodies bound to PMNs in 38 percent of these 47 patients. Since only about one in three patients with febrile transfusion reactions had detectable IgG anti-PMN antibodies, fever may be caused by lysis of leukocytes other than PMNs.     

A nuclear antigen associated with cell proliferation and blast transformation

A nuclear antigen associated with cell proliferation (proliferating cell nuclear antigen-PCNA) and blast transformation is recognized by autoantibodies in the sera of some patients with systemic lupus erythematosus. This autoantibody is a precipitating antibody and also reacts in immunofluorescence, staining the nucleoplasm of proliferating and blast-transformed cells. The autoantibody was used as a reagent to determine the distribution of PCNA in a synchronized continuous B lymphoid cell line (WiL-2) and in mitogen-induced blast-transformed lymphocytes. In WiL-2 cells, PCNA was detected as speckled nucleoplasmic staining in G1, S, and G2 phases of the cell cycle. In addition, during late G1 and early S phases, PCNA was also detected in the nucleolus. During mitogen-induced blast transformation of lymphocytes, PCNA was noticed in the nucleolus before the initiation of DNA synthesis and later became nucleoplasmic with disappearance of nucleolar staining. These studies demonstrate that the relationship of PCNA to proliferation and blast transformation may be associated with events related to DNA synthesis in these cells.     

Early endosome antigen. 1: An autoantigen associated with neurological diseases.

BACKGROUND: We have identified 36 human sera sent for autoantibody analyses that produce a unique vesicular staining pattern of the cytoplasm of tissue culture cells. The purpose of this study was to identify the autoantigens that are recognized by the sera that produce this staining pattern and determine if the patients have common clinical features. METHODS: A serum from one of the patients (MS) with rapidly progressive demyelinating polyneuropathy was used to isolate a approximately 4.5 kb cDNA insert from a HeLa expression library. The purified cDNA (MS-5.1) was characterized by a poly A tail and an open reading frame that encoded 1329 amino acids. The derived amino acid sequence was found to be 99% identical to a 180 kd peripheral endosomal protein named early endosome antigen (EEA1). RESULTS: Antibodies from rabbits immunized with the recombinant protein and the prototype human serum produced an identical distinctive speckled cytoplasmic staining pattern. These sera also precipitated the in vitro translated recombinant protein and reacted with the isolated recombinant protein in a Western immunoblot. Of the 36 sera that produced an identical staining pattern as the prototype and immune rabbit sera, 8 (22%) had IgG antibodies that recognized the recombinant EEA1 protein when tested by immunoblotting and immunoprecipitation assays. Of the 8 patients with anti-EEA1 antibodies 4 were females, 4 were males, and the mean age was 69 years (range 48 to 86 years). CONCLUSIONS: Diagnoses included: polyneuropathy, lower motor neuron disease, pigmented retinitis, seronegative polyarthritis, interstitial pulmonary fibrosis, Raynaud's phenomenon, Wegener's granulomatosis, and proteinuria. Three of the eight patients with EEA1 autoantibodies died within 1 year after EEA1 antibodies were identified.