Robert L. Rubin

Antinuclear antibodies (ANAs): diagnostically specific immune markers and clues toward the understanding of systemic autoimmunity

The convergence of studies in the clinical and basic sciences has resulted in the definitive identification of many intracellular antigens which are the targets of autoantibodies in patients with systemic lupus erythematosus, scleroderma, dermatomyositis/polymyositis, Sjogrens syndrome, mixed connective tissue disease, and drug-induced autoimmunity. Some of this new knowledge includes the identification of the Sm and RNP antigens as ribonucleoprotein particles involved in splicing of precursor messenger RNA, Scl-70 as DNA topoisomerase I, proliferating cell nuclear antigen as auxiliary protein of DNA polymerase delta, and certain antigens in myositis as aminoacyl transfer RNA synthetases. This information confirms, at a molecular level, the presence of specific profiles of autoimmune responses so that autoantibodies can be used in clinical medicine as diagnostically useful immune markers. In addition the data give compelling reasons to consider that certain autoimmune diseases are antigen-driven. Many auto-antibodies have the interesting feature of recognizing epitopes on the antigens which are active or functional sites of the molecule. It is suggested that the data provide clues to the nature of the intracellular particle initiating the immune response and may help to elucidate some of the early mechanisms of the autoimmune process.

Serum deoxyribonuclease I and clinical activity in systemic lupus erythematosus

SummarySerum deoxyribonuclease I (DNase I) activity in systemic lupus erythematosus (SLE) patients was shown to be lower than that of healthy laboratory personnel, rheumatoid arthritis, and scleroderma patients (P<0.001). The decrease in DNase I activity in SLE sera was not due to the effect of various autoantibodies or to heat labile DNase I inhibitor. A relationship between serum DNase I activity and active SLE was demonstrated. Patients with active lupus nephritis had the lowest levels of enzymatic activity.

Subnucleosome structures as substrates in enzyme-linked immunosorbent assays.

Histone preparations preserving the tertiary and quaternary structure of histone-histone complexes and histone-DNA complexes, as well as individual histones, were isolated or reconstituted. Various parameters were tested in order to determine the suitability of these complexes for use as substrates in enzyme-linked immunosorbent assays. The protein concentration required to saturate the solid phase was determined, and the amount of bound protein was quantified by the micro-bicinchoninic acid protein assay. In addition, the relative DNA content of solid phase antigen was measured by the binding of monoclonal anti-native DNA antibodies. Prototype sera representing different disease groups produced reproducible and unique patterns of reactivity on the panel of antigens, demonstrating the lack of substantial assay bias. Two substrates, the H2A-H2B dimer and the H2A-H2B-DNA complex, both appear to be oriented in a random manner on the solid phase, leaving a number of different epitopes exposed to the solution. This novel set of histone antigens can now be used to define the specificity of anti-histone antibodies in relation to the quaternary structure of chromatin.

An improved ELISA for anti-native DNA by elimination of interference by anti-histone antibodies

We evaluated an enzyme-linked immunosorbent assay (ELISA) for antibodies to native DNA (nDNA) in which protamine was used to link DNA to polystyrene. Elevated anti-nDNA was largely restricted to patients with systemic lupus erythematosus (SLE), and within this group good correlation between ELISA and the ammonium sulfate assay was obtained. However, substantial background immunoglobulin binding to protamine coated wells was commonly observed, and it was necessary to subtract this activity from each anti-DNA determination. Many of the SLE sera also contained anti-histone antibodies, and this antibody activity showed significant correlation with the binding to protamine. In contrast, methylated bovine serum albumin (mBSA) did not bind anti-histone antibodies and provided a substrate for coupling nDNA to polystyrene. This modified ELISA allowed the quantitation of antibodies to native DNA without the simultaneous binding of anti-histone antibodies.

Reactivity of anti-histone antibodies induced by procainamide and hydralazine☆

Abstract Sera from patients with procainamide (Pr)- or hydralazine (Hy)-induced lupus were examined for anti-histone antibodies by an immunofluorescence assay using histonerecostituted mouse kidney sections and by a solid-phase radioimmunoassay using polystyrene tubes coated with purified histones. Distinct differences were observed between Pr sera and Hy sera. Pr sera were uniformly positive in the immunofluorescence assay on histone-reconstituted tissue sections, but all Hy sera were negative. In solidphase radioimmunoassay, both Pr and Hy sera showed anti-histone activity with Pr sera demonstrating strong reactions with H2A-H2B histone complex but Hy sera demonstrating weak reactions. The difference in the immunofluorescence assay could in most but not all instances be attributed to this difference in anti-H2A-H2B activity. Antibodies were not detected to DNA or to nonhistone nuclear antigens Sm, nuclear ribonucleoprotein, and SS-B La . The results indicate that both Pr and Hy induce antibodies to histones and that anti-histone antibodies induced by Pr differ both quantitatively and qualitatively from those induced by Hy.

HLA-DR2 association with excessive somnolence in narcolepsy does not generalize to sleep apnea and is not accompanied by systemic autoimmune abnormalities.

Recent reports that nearly all patients with narcolepsy have the HLA-DR2 phenotype suggest that autoimmunity may underly the etiology or pathogenesis of this disorder. Of 11 narcoleptic patients in the present study, 9 were HLA-DR2, confirming the strong association with this class II antigen but indicating that this is not an obligatory phenotype. In contrast only 3/10 patients with sleep apnea were HLA-DR2, suggesting that this form of excessive somnolence has a different etiopathogenesis. Significant levels of rheumatoid factor, antinuclear antibodies or autoantibodies to native DNA, denatured DNA, histones, Sjogrens syndrome B antigen, or Smith antigen were undetectable in sera from narcoleptic patients. Antibodies to rodent brain, primate brain stem, and neurocytotoxic antibodies were also not found. These results along with the absence of laboratory signs and clinical features of a systemic inflammatory process indicate that if narcolepsy is an autoimmune disease, the underlying lesion or pathologic condition may be confined to the central nervous system.

IgG antibodies to the histone complex H2A-H2B characterize procainamide-induced lupus☆

Patients treated with procainamide and other drugs commonly develop antinuclear antibodies and occasionally symptoms of lupus erythematosus. However, the pathological events which lead to clinical symptoms in some patients but only abnormal serology in others have not been established. The present study examines the incidence, amount, immunoglobulin class, and antigen-binding specificity of anti-histone and anti-denatured DNA (anti-dDNA) antibodies in three groups of patients. These comprised a prospective study of patients treated with procainamide, patients with clinical drug-induced lupus symptoms, and a group undergoing therapy for many years without any symptoms. Procainamide elicited IgG and IgM anti-dDNA antibodies concordantly. Anti-histone IgM antibodies also appeared de novo during this period but IgG anti-histone antibodies were detected less frequently. Asymptomatic patients tended to have an antibody profile consisting of highly elevated anti-dDNA, IgM antibodies reactive with all histones and IgG antibodies specific for only one or two histone classes. In contrast symptomatic patients usually had little anti-dDNA or antibodies to individual histones but had pronounced IgG antibodies to the histone complex H2A-H2B. This unique antibody was characteristics of procainamide-induced lupus and was not detected in patients whose disease was induced by hydralazine. Anti-(H2A-H2B) decreased after procainamide was discontinued, concomitant with subsidence of symptoms. The finding that autoantibodies elicited by procainamide in patients with lupus symptoms have a characteristic immunoglobulin class and specificity may be of pathogenic significance and suggests that patients susceptible to procainamide-induced lupus have a unique immune response. In addition, this information could be of diagnostic value in predicting which procainamide-treated patients will develop overt symptoms of lupus.

Serologic changes during. Induction of lupus-like disease by procainamide

Procainamide-induced lupus is a well-recognized syndrome, but the events leading up to clinical symptoms are obscure. In the present study, serologic changes in a 69-year-old man were monitored during his treatment with procainamide and after discontinuation of procainamide because of symptoms of drug-induced lupus. Antihistone antibodies of unique specificity and in vivo complement activation were detected after one year of procainamide therapy during a period prior to development of significant clinical symptoms. Antihistone antibodies and complement activation substantially increased during a full-blown episode of lupus-like symptoms. Progressive return to normal laboratory findings occurred after procainamide was discontinued. The antihistone/complement profile may be useful in the diagnosis of drug-induced lupus and warn of impending clinical deterioration in patients with minimal symptoms.

Pseudoautoimmunity in normal mice : anti-histone antibodies elicited by immunization versus induction during graft-versus-host reaction

Native preparations of evolutionarily conserved intracellular macromolecules are generally nonimmunogenic when injected in soluble form. However, vigorous immune responses were observed when common autoantigens such as histones, DNA or Sm antigen, or homologous liver homogenate were noncovalently coupled to latex beads prior to injection into mice. Antibody response to histone beads displayed immunologic memory and required a functional thymus, suggesting that T-helper cells were involved. However, bead-elicited autoantibodies could be distinguished from true autoantibodies in that they reacted with denatured, minor, or foreign components of the preparations or to regions unexposed in the native form of the immunogen. This response contrasted with spontaneously arising autoantibodies accompanying graft-versus-host (GVH) disease in the same strain of mice which preferred native nucleoprotein conformations within nuclei, chromatin, or DNA-histone complexes. Histone beads elicited antihistone antibodies displaying a sustained IgM isotype in contrast to spontaneously arising autoantibodies in GVH disease which were predominantly IgG. These studies demonstrate that immunization with autoantigens does not usually elicit true autoantibodies and suggest that lymphocyte populations responsible for pseudoautoimmune responses are different from autoantibody-producing cells. We speculate that if autoimmunity is driven by particulate forms of in vivo self-materials, additional factors are required for breaking the natural tolerance to native conformations within the immunogen.

Autoantibodies to native myeloperoxidase in patients with pulmonary hemorrhage and acute renal failure

Sera from 245 patients were screened by indirect immunofluorescence for perinuclear/nuclear staining (P-ANCA) of ethanol-fixed neutrophils, a staining pattern which is associated with the presence of antibodies to myeloperoxidase. Using immunoblot and immunoprecipitation techniques on 15 P-ANCA-positive sera, 13 patients demonstrated antibody to purified or native myeloperoxidase but not to denatured myeloperoxidase. In patients with P-ANCA, the most frequent reason for medical attention was hemoptysis (8/13; 62%). Of the 15 sera with P-ANCA, acute renal failure was identified in 9 patients (60%). Five patients (33%) had both. All patients (eight of eight) with hemoptysis had antibodies which bound functional MPO as compared to three of seven P-ANCA-positive patients without hemoptysis (P<0.001), suggesting that antibodies which recognize conformational sites on native myeloperoxidase occur in a subgroup of patients with alveolar hemorrhage as their presenting clinical sign. These findings may provide insight into the disease process associated with P-ANCA. We further identify a subgroup of patients with a severe pulmonorenal syndrome and antibodies recognizing native myeloperoxidase.