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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 bio-chemically 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.

Objective: To determine the specificity of anti-CD45 autoantibodies in systemic lupus erythematosus (SLE) for native CD45 and for CD45 expressed by T cells and B cells, and at different stages of cellular activation., Methods: CD45 purified from different types of lymphocytes was examined by immunoblotting with sera from patients with SLE. Indirect immunofluorescence experiments were performed with purified anti-CD45 autoantibodies., Results: IgM anti-CD45 autoantibodies in SLE recognize native CD45 expressed on the surface membrane of viable lymphocytes and CD45 purified from activated peripheral T cells and certain T cell lines, but not CD45 purified from B cells or resting peripheral T cells. The presence or absence of reactivity is independent of the individual isoforms expressed., Conclusion: Recognition of CD45 by IgM antilymphocyte autoantibodies in SLE varies with the lineage and state of activation of the lymphocyte target. This pattern of reactivity is consistent with autoantibody specificities involving CD45 glycoforms, rather than CD45 isoforms.

Normal individuals possess low levels of autoantibodies specific for certain peptide defined regions of T-cell receptor (Tcr) variable regions, particularly CDR1 and Fr3. These regions are predicted to be exposed on the surface of the native molecule and, by analogy and comparison with immunoglobulins, correspond to public idiotype determinants. The anti-Tcr idiotype antibodies appear to be ubiquitous and we propose that they play a role in the regulation of T-cell function. To delineate the parameters of expression of these antibodies, we characterized anti-Tcr antibody activity in normal individuals, in those suffering from the autoimmune diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), and in patients with non-autoimmune arthritis (osteoarthritis) as a disease control. There were significant increases in autoantibody levels in the autoimmune patients. There was also variation in isotype and the particular variable regions recognized. IgM autoantibodies directed against a few peptide defined determinants were elevated in RA, whereas SLE patient sera showed high levels of IgG binding to a broad spectrum of Tcr peptides.

The fetus is a natural allograft that is protected from immunologic rejection by a complex set of structural and regulatory mechanisms. We determined whether healthy pregnant women differed significantly from healthy non-pregnant controls in their capacity to produce autoantibodies to defined antigenic determinants of the alpha/beta T-cell receptor. Although controls and pregnant women expressed comparable levels of autoantibodies against an intact recombinant T-cell receptor containing the complete V alpha/V beta structures, analysis of comparative reactivity against individual peptide segments of the molecules, indicated enhanced reactivity to regions corresponding to the CDR1 of the alpha chain and to the Fr3 of the variable region of the beta chain. A major difference was noted by increased reactivity of IgG autoantibodies of pregnant women to peptides corresponding to the "switch" region joining the variable and constant domains. This was noted with both the Tcr alpha and beta chains and was directed against highly conserved determinants within these molecules. Antibodies to this region are lacking in the non-pregnant controls. It is possible that autoantibodies directed against conserved regions of the T-cell receptor might function in the suppression of T-cell reactivity of fetal determinants.

A 24-year-old black woman developed polymyositis with autoantibodies to threonyl-tRNA synthetase in the 2nd trimester of her 3rd pregnancy. This was complicated by fetal loss and the development of severe relapsing myositis resistant to corticosteroid and azathioprine therapy. These features were also common in other cases in the literature. Antisynthetase antibodies had not been reported in myositis occurring during pregnancy and may be of interest regarding the pathogenesis of inflammatory myopathy complicating pregnancy.

Patients with SLE develop IgM autoantibodies to different isoforms of CD45, the major surface membrane protein tyrosine phosphatase on lymphocytes and other nucleated hemopoietic cells. Because such autoantibodies could have a potential role in the development of immune dysfunction in this disorder, we performed a series of experiments to characterize their antigenic specificity further. Blots of recombinant E. coli fusion proteins encoded by exons 3-7 of the p220 and p180 isoforms were uniformly non-reactive with SLE IgM, suggesting that anti-CD45 autoantibodies in SLE are directed against conformational and/or carbohydrate epitopes, rather than linear polypeptide epitopes. This issue was examined further using chemically and enzymatically modified CD45 purified from T cells by lectin affinity chromatography as substrates. Treatment of CD45 with 25 mM sodium-m-periodate, sufficient to abrogate binding to various lectins, abolished the reactivity with SLE anti-CD45 autoantibodies. On the other hand, digestion of CD45 with neuraminidase enhanced the binding of anti-CD45 autoantibodies from some of the SLE sera. This result probably reflects decreased steric hindrance or charge repulsion because the binding of mouse monoclonal antibodies directed against linear polypeptide epitopes of CD45 was similarly enhanced. Digestion of CD45 with N-glycosidase F had no effect on autoantibody staining. Taken together, these data suggest that IgM anti-CD45 autoantibodies in SLE recognize non-sialylated carbohydrate determinants in the highly O-glycosylated polymorphic domains of CD45.

IgM autoantibodies to nucleolin and histone H1 are strongly associated in the serum of patients with systemic lupus erythematosus. IgM eluted from immobilized nucleolin specifically stained histone H1 blotted to nitrocellulose; conversely, IgM eluates prepared from immobilized histone H1 stained nucleolin blots. We conclude that the linkage of anti-nucleolin and anti-histone H1 autoantibodies in SLE is due, at least in part, to immunologic cross-reactivity between these two autoantigens, which share certain similar structural features.

Autoantibodies to surface antigens on lymphocytes and other cells of the immune system may contribute to the development of immunoregulatory and other cellular immune abnormalities in patients with active systemic lupus erythematosus. Of special interest in this respect are autoantibodies to CD45 (leukocyte-common antigen, T200), a plasma membrane protein tyrosine phosphatase implicated in the regulation of lymphocyte functional activity, including cytotoxicity, proliferation, and differentiation.

Unselected sera from patients with various rheumatic, inflammatory bowel, and autoimmune skin diseases (n = 268) were examined against human cell lysate by immunoblotting procedures, to determine the prevalence of autoantibodies to stress proteins (heat-shock proteins) hsp60 (homolog of Escherichia coli groEL and mycobacterial 65K antigens), hsp73, and hsp90. Using standard, sensitive and specific assay conditions, IgG and IgM autoantibodies to these stress proteins were not demonstrable, or were detected infrequently, in sera from control subjects (n = 36) and from patients with rheumatoid arthritis, Sjögren's syndrome, ankylosing spondylitis, Reiter's syndrome, systemic lupus erythematosus, and systemic sclerosis. Autoantibodies to hsp60 were relatively more common (greater than or equal to 20% of sera) in patients with mixed connective tissue disease, polymyositis/dermatomyositis, psoriatic arthritis, inflammatory bowel disease, epidermolysis bullosa acquisita, and bullous pemphigoid. Anti-hsp73 autoantibodies were detected in 20% or more of the sera from patients with Lyme disease and ulcerative colitis. Taken together, these data extend the spectrum of autoimmune and inflammatory diseases in which humoral anti-stress protein autoreactivity develops. However, the paucity of humoral autoreactivity to stress proteins in patients with systemic lupus erythematosus and rheumatoid arthritis argues against a direct role of anti-stress protein autoantibodies in the pathogenesis of these disorders.

The 110-kDa intracellular phosphoprotein (110K) described previously by this laboratory as a common IgM autoantigen in SLE and certain other systemic autoimmune disorders and viral infections is identified as nucleolin in the present investigation. Using rabbit antiserum to rat nucleolin as a probe, IgM autoantibody-reactive 110K co-migrated with human lymphocyte nucleolin in one- and two-dimensional immunoblots. Rabbit anti-nucleolin also specifically depleted autoreactive 110K from detergent lysates of human cells. Because nucleolin shares amino acid sequence similarity and/or forms dynamic particles with other prominent autoantigens, the present observation raises the possibility that the nucleolin/anti-nucleolin system may be of special significance for the development of humoral autoreactivity to nuclear Ag.

Nearly one-third of IgM antilymphocyte autoantibody-positive sera from patients with systemic lupus erythematosus (SLE) contain IgM antibodies to one or more 180-220-kD molecules (p180, p190, p205, and p220) in blots of glycoproteins purified from T cells by wheat germ agglutinin affinity chromatography. Identity of these IgM targets with multiple isoforms of CD45 was established by their specific depletion from T cell glycoproteins by immunoprecipitation with T191, a monoclonal antibody (mAb) that reacts with an epitope common to all CD45 isoforms. Although the anti-CD45 autoantibodies recognize higher molecular weight isoforms primarily, antigenic specificity in this system is quite heterogeneous and includes multiple distinct CD45 isoforms on different types of T cells that are, at least in part, different from those reactive with mAbs 2H4 and UCHL-1. Because CD45 is a major membrane protein tyrosine phosphatase that plays a critical role in antigen-induced T cell activation, the present data may be relevant to some of the antilymphocyte antibody-mediated immunologic abnormalities that characterize SLE and related autoimmune diseases.

Sera from patients with systemic lupus erythematosus frequently contain IgM antibodies to glycoproteins of Mr 46,000 and approximately 200,000 isolated from nonionic detergent lysates of mature T cells by affinity chromatography with solid-phase wheat germ agglutinin. Autoantibodies of this specificity correlate strongly with the presence of IgM anti-T cell autoantibodies, as determined by independent indirect immunofluorescence and complement-dependent microcytotoxicity assays, and are specifically absorbed by incubation of patient serum with viable T cells. Collectively, the data suggest that gp46 and, to a lesser extent, gp approximately 200 represent major targets of IgM antilymphocyte autoantibodies in systemic lupus erythematosus.

Utilizing nonionic detergent lysates of human lymphoid and non-lymphoid cells as substrate, IgM and/or IgG antibodies to a 110-kDa/isoelectric point 5.4 phosphoprotein (110K) was demonstrated in serum from patients with SLE or certain other systemic autoimmune disorders by immunoblotting and immunoprecipitation. Ig of this specificity was not demonstrable in serum from normal individuals, but, in a limited survey, was detected in serum from patients with acute hepatitis A or infectious mononucleosis. 110K shares a number of properties with nucleolin, i.e., identical Mr and isoelectric point, localization in both the nucleus and the cytosol, increased expression in rapidly dividing cells, and shown to be distinct from already defined autoantigens of similar size, i.e., topoisomerase I, PM-Scl, and RNA polymerase I. Because 110K could bind denatured DNA, as demonstrated by its specific absorption by DNA-cellulose and by its reactivity with monoclonal anti-ssDNA antibody in the presence of denatured DNA, special efforts were made to distinguish reactivity of pre-formed DNA/anti-DNA complexes in SLE serum from that due to specific anti-110K autoantibodies. Although binding to 110K could be mediated by DNA and anti-DNA in some SLE sera, the accumulated evidence supports the existence of a major new autoantibody system in SLE, other autoimmune diseases, and certain virus infections.

Sera from 20 patients with systemic lupus erythematosus (SLE) and active central nervous system (CNS) dysfunction were examined by indirect immunofluorescence for antibodies to neuronal membrane determinants. Warm-reactive IgG antibodies were demonstrable in 82% (9/11) of patients with clinical evidence for seizures or diffuse CNS disease, but these antibodies generally were absent in non-CNS SLE sera or when focal neurologic deficit or psychosis was the primary CNS manifestation. Cold-reactive antibodies of the IgM class were equally prevalent in patients with or without CNS disease and appeared to be more directly correlated with extra-CNS systemic illness. Absorption experiments with lymphocytes, brain homogenate, and various other tissues suggested a predominant brain-specificity for IgG antibodies and partial lymphocyte cross-reactivity for IgM antibodies. Interpretations of this special association between IgG anti-brain antibodies and diffuse CNS dysfunction in SLE are discussed.

IgG anti-T cell autoantibodies are common in SLE serum, react preferentially with activated lymphocytes, and exert early-phase inhibitory effects on antigen-induced T cell proliferation. Little is known about the target molecules in this system, however, because the low titer and low avidity of the most interesting antibodies limit their utility in conventional immunoprecipitation analyses. Therefore, Western blotting was used to demonstrate binding of IgG in anti-T cell antibody-positive SLE sera to four surface membrane molecules shared by peripheral T cells and HSB-2 cells. Molecules of Mr 90,000 and 55,000 were particularly reactive: each target was stained by IgG anti-lymphocyte antibodies in 11 patient sera (approximately 85%) in the panel. Targets of Mr 37,000 and 105,000 were encountered less frequently (six of 13 and one of 13 patients, respectively). It is unlikely that alloantibodies contributed to the staining patterns observed because reactivity with the four targets was consistently present when cell preparations from multiple unrelated donors were examined. The target molecules were localized to the plasma membrane by whole cell absorption/elution experiments, by the failure of chromatin (DNA/histone) to absorb antibodies to these antigens, and through the use of purified membranes as substrate for Western blotting. With the possible exception of the 105,000 Mr molecule, which is a major target in the IgM anti-T cell antibody system, evidence for the existence of neoantigens as a basis for increased reactivity of SLE IgG with activated T cells was not obtained. The identity of the IgG antibody-reactive molecules with respect to known T cell antigens was not determined, although evidence against the existence of antibodies to Tac (IL 2 receptor) and the transferrin receptor was obtained in monoclonal antibody pre-clearing experiments. Nonetheless, the observation that a limited number of major IgG autoantibody target antigens on activated peripheral T cells are shared by HSB-2 cells, a primitive T cell line expressing few of the differentiation antigens characteristic of mature T cells, should provide a basis for more definitive characterization of antigens in this system in the future.

Patients with systemic lupus erythematosus frequently develop antilymphocyte antibodies as measured by complement-dependent cytotoxicity and immunofluorescence assays. Highest titres of both of the major IgM and IgG classes occur during phases of active disease, and their presence is associated with essentially the entire spectrum of immune system functional abnormalities in this disorder. While the full range of antibody specificities requires further clarification, antibodies to many discrete lymphocyte populations have been described, including B cells, T cells, and T cell subsets. Antibodies to T cell subsets are of special interest because of their relationship with subset depletion in vivo, and their capacity to reproduce, through effects on normal cells in vitro, the same types of immunoregulatory abnormalities characteristic of lymphocytes isolated from patients with SLE. Suppressor/inducer and suppressor/effector T cells appear to be the main targets in this regard. Antibodies specific for activated T lymphocytes exist as well, and this type has the unusual property of interfering with events operant in production of/response to interleukin-2, a critical step controlling the expansion of specifically-reactive T cells and the induction of other lymphokines. In addition to complement-mediated lysis and antibody-dependent cell-mediated cytotoxicity, anti-lymphocyte antibodies have the potential to influence immune system function by several non-cytotoxic mechanisms, including surface antigen modulation and ligand/receptor triggering. Despite the large amount of data which has been accumulated concerning the cell type specificity and functional effects of anti-lymphocyte antibodies in SLE, little is known about the nature of the surface membrane molecules with which they react. Application of cell cloning and molecular biology technology should rectify this deficiency in the near future. Although it is likely that antilymphocyte antibodies are of relevance to immune system pathophysiology in SLE, it remains to be determined whether these interesting antibodies reflect secondary events, or have some more fundamental significance.

Shedding of cell-surface antigens that react with anti-lymphocyte autoantibodies in systemic lupus erythematosus (SLE) is well-recognized, but the nature of such molecules is unknown. The present investigation demonstrates the rapid shedding of three IgG antibody target molecules of Mr 55,000, 37,000, and approximately 32,000 from the surface of mitogen-activated peripheral T cells during brief incubation at 37 degrees C. Sera lacking IgG anti-lymphocyte antibodies stained none of the three antigens. Absorption of antibody-positive sera with viable HSB-2 cells, a primitive T-cell line lacking HLA antigens and many CD antigens characteristic of mature peripheral T cells, eliminated staining of the shed molecules. These data delineate the number and estimated molecular mass of anti-lymphocyte autoantibody target molecules that are shed from the surface of T cells, and provide further insight into potential mechanisms by which anti-lymphocyte antibodies contribute to the pathogenesis of SLE and related disorders.

Serum from patients with systemic lupus erythematosus (SLE) frequently contain IgM and IgG autoantibodies to the constitutively expressed 73-kD/pI 5.5 member of the hsp70 family of heat shock proteins, as determined by one-dimensional (SDS-PAGE) and two-dimensional (IEF/SDS-PAGE) immunoblotting, and by solid-phase SLE Ig immunoprecipitation experiments using hsp70 protein-specific mAbs as probes. Autoantibodies to hsp70 also were detected in a minority of sera from patients with other rheumatic or viral diseases, but not in normal sera. These data may provide additional insight into etiologic and pathophysiologic mechanisms in this and related autoimmune disorders.

In addition to previously described cold-reactive IgM anti-lymphocyte antibodies maximally cytotoxic for resting cells at 15 degrees C, sera from patients with systemic lupus erythematosus (SLE) were found to contain a new type of antibody preferentially reactive at physiologic temperatures with mitogen-activated lymphocytes. This antibody lacked specificity for unstimulated lymphocytes, and was shown to be of the IgG class both by indirect immunofluorescence and in immunochemical experiments. Certain SLE sera also contained IgG antibodies with the capacity to develop plaques with mitogen-activated T lymphocyte preparations used in a reverse hemolytic plaque assay, indicating reactivity with products released by activated cells. The elimination of the ability of SLE sera to develop plaques after absorption with viable mitogen-stimulated lymphocytes, but not with resting cells, suggested that these antibodies were directed toward activation "neoantigen(s)" shed from the cell surface membrane. Surface membrane phenotype analyses performed by using a variety of monoclonal antibody reagents indicated that the plaque-forming cells (PFC) detected with SLE sera were activated T lymphocytes not restricted to single OKT4+, OKT8+, or Ia antigen+ subpopulations. Essentially all PFC expressed transferrin receptors. The present data raise the possibility that certain of the interesting effects of anti-lymphocyte antibodies on immunologic function in SLE may be mediated by interactions of these new type(s) of antibodies with activated lymphocytes or their products, rather than through blocking or depletion effects on resting precursor cells.

The subclass distribution of anti-Sm antibodies in the serum of MRL/Mp-Ipr/Ipr and MRL/Mp- +/+ mice was investigated with a sensitive ELISA technique. In both strains, but particularly in the Ipr mice, anti-Sm antibodies were predominantly of the IgG2a isotype. This preponderance was not an artifact of the sensitivity or specificity of the subclass-specific anti-Sm ELISA, nor did it reflect the subclass distribution of total serum IgG. A plaque-forming cell assay for anti-Sm antibodies also showed a predominance of the IgG2a isotype, indicating that the serum findings could not be explained by differential catabolism of IgG subclasses. Finally, antibodies to double-stranded DNA, as detected by the Crithidia luciliae assay, did not show a restricted subclass distribution. The isotype expression of anti-Sm antibodies in MRL mice must reflect in vivo mechanisms regulating the production of these autoantibodies. The IgG2a restriction in particular suggests an important in vivo role for T cells.

Three cell lymphocyte antigens of m.w. 55,000, 70,000, and 105,000 to 110,000 were identified by Western blotting as targets of IgM autoantibodies in serum from a group of 49 patients with systemic lupus erythematosus. The 55- and 70-kDa antigens were well expressed on unstimulated peripheral T cells, whereas the 105- to 110-kDa target was demonstrable only on mitogen-activated T cells and lymphoblastoid T cell lines. Localization of these molecules to the plasma membrane was established by cytoabsorption experiments in which IgM antibody staining of blotted antigens was specifically absorbed from systemic lupus erythematosus serum during 4 degrees C incubations with intact lymphocytes, and by their detection in purified lymphocyte plasma membranes. While the identity of these target antigens vis a vis known surface determinants was not defined, their expression on peripheral T cells from multiple donors and on cell lines of both undifferentiated (HSB-2) and phenotypically mature (Jurkat; HUT 78) types excluded alloantigens, major histocompatibility complex-encoded determinants, and most T cell differentiation antigens as candidates in this regard. Expression of the IgM autoantibody targets on HSB-2 cells argues against discrete T subset specificities as well. IgM reactivity with the 55-, 70-, and 105- to 110-kDa antigens by blotting was highly correlated with antilymphocyte antibody activity in complement-dependent cytotoxicity assays (Fisher's p less than 0.001), and paralleled flow microfluorimetric and microcytotoxicity quantitation of IgM antibody activity in serial observations of individual patients studied during different phases of disease activity. Taken together, these data suggest that IgM lymphocytotoxic antibodies in systemic lupus erythematosus are directed predominantly against a limited number of non-T cell subset-specific antigens.