Clues to the etiology of autoimmune diseases through analysis of immunoglobulin genes

The role of autoantibodies in the etiology of autoimmune diseases remains unclear. However, an examination of the sequences of these autoantibodies can be informative. Antibody sequences that violate constraints normally imposed during ontogeny and during development point to a failure of regulation. The existence of clonally related sequences indicates that production of these antibodies may frequently be driven by self-antigen. A better understanding of the mechanisms that normally constrain the composition of the antibody repertoire and of the nature of the inciting and/or driving antigens may yield new insights into both the pathogenesis and potential treatment of these crippling diseases.     

Inhibition of caspase-3-mediated poly(ADP-ribose) polymerase (PARP) apoptotic cleavage by human PARP autoantibodies and effect on cells undergoing apoptosis

Autoantibodies directed to nuclear antigens are serological hallmarks of autoimmune rheumatic diseases such as systemic lupus erythematosus. Although much more is known about the molecular identity and functions of targeted self-antigens, with few exceptions, evidence that autoantibodies to these targets have a particular function and contribute directly to the pathological process is lacking. Here we show that human autoantibodies reacting with the zinc fingers of poly(ADP-ribose) polymerase involved in the recognition of damaged DNA totally prevent the cleavage of poly(ADP-ribose) polymerase by caspase-3, a process that normally occurs during early apoptosis. Furthermore, these antibodies, which are frequent in certain autoimmune rheumatic and bowel diseases, affect the characteristic features of apoptosis and increase cell survival ex vivo. This new observation is important, because failure to remove autoimmune or abnormal cells can give rise to prolonged autoimmune stimulation and tumor formation.     

V region sequences of an idiotypically connected family of pathogenic anti-DNA autoantibodies

The H and L chain V region sequences of nine anti-DNA mAb that are representative of a pathogenic population of autoantibodies produced by the nephritis prone (SWR x NZB)F1 (SNF1) mice, were determined. These nine anti-DNA autoantibodies were idiotypically connected members of a cross-reactive Id family called the Id564 cluster. Moreover, these autoantibodies were all cationic in charge, IgG2b in isotype, and their H chain C regions had the normal SWR parent's allotype. Although derived from two different SNF1 animals, these pathogenic autoantibodies possessed highly homologous Leader-VH sequences that could account for their idiotypic cross-reactivity. Furthermore, the VH region sequences of these anti-DNA antibodies contained numerous basic residues that could impart their cationic charge. The Leader-VH sequences of these autoantibodies were also highly homologous to that of an anti-NP antibody-related germ-line gene of C57BL/6 mice, called VH-23. Among these nine pathogenic autoantibodies, three sets of clonally related anti-DNA antibodies could be identified. Thus the Id564 cluster of cationic anti-DNA autoantibodies of SNF1 mice are encoded by highly related VH genes, and this idiotypically connected population of pathogenic autoantibodies are selected to undergo an oligoclonal expansion in the lupus-prone SNF1 mice.     

Autoreactive T‐cell receptor (Vβ/D/Jβ) sequences in diabetes are homologous to insulin,glucagon, the insulin receptor,and the glucagon receptor

The hypervariable (Vβ/D/Jβ) regions of T‐cell receptors (TCR) have been sequenced in a variety of autoimmune diseases by various investigators. An analysis of some of these sequences shows that TCR from both human diabetics and NOD mice mimic insulin, glucagon, the insulin receptor, and the glucagon receptor. Such similarities are not found in the TCR produced in other human autoimmune diseases. These data may explain how insulin, glucagon, and their receptors are targets of autoimmunity in diabetes and also suggest that TCR mimicking insulin and its receptor may be targets of anti‐insulin autoantibodies. Such intra‐systemic mimicry of self‐proteins also raises complex questions about how “self” and “nonself” are regulated during TCR production, especially in light of the complementarity of insulin for its receptor and glucagon for its receptor. The data presented here suggest that some TCR may be complementary to other TCR in autoimmune diseases, a possibility that is experimentally testable. Such complementarity, if it exists, could either serve to down‐regulate the clones bearing such TCR or, alternatively, trigger an intra‐immune system civil war between them. Copyright © 2008 John Wiley & Sons, Ltd.     

Proteomics-based identification of autoantibodies in the sera of healthy Chinese individuals from Beijing

The identification of panels of tumor antigens that elicit an antibody response may have utility in cancer screening, diagnosis and in establishing prognosis. However, autoantibodies normally exist in sera of healthy individuals and are enormously diversified. To explore the reservoir of autoantibody in healthy population, we performed a proteomics investigation of autoantibody profiles in the sera of 36 healthy Chinese individuals from Beijing, which may provide valuable reference information to the identification of disease-specific autoantibodies. The results showed that autoantibody profiles varied individually, but some autoantibodies were identified at a high frequency in the healthy population. The autoantibodies against alpha-enolase and those against heterogeneous nuclear ribonucleoprotein L were positive in more than 50% of the sera samples. The autoantibodies identified in more than 20% of samples included those against annexin II, F-actin capping protein beta subunit and calreticulin. Some of these autoantibodies have been previously reported to be involved in autoimmune conditions and cancers. Autoantibodies in the healthy population are important as a foundation from which disease-specific autoantibodies can be defined. Thus our report on autoantibodies in healthy individuals may be useful as a reference for defining new autoantibody biomarkers.     

Fragmentation of Golgi complex and Golgi autoantigens during apoptosis and necrosis

Anti-Golgi complex autoantibodies are found primarily in patients with Sjögren's syndrome and systemic lupus erythematosus, although they are not restricted to these diseases. Several Golgi autoantigens have been identified that represent a small family of proteins. Common features of all Golgi autoantigens appear to be their distinct structural organization of multiple α-helical coiled-coil rods in the central domains flanked by non-coiled-coil N-termini and C-termini, and their localization to the cytoplasmic face of Golgi cisternae. Many autoantigens in systemic autoimmune diseases have distinct cleavage products in apoptosis or necrosis and this has raised the possibility that cell death may play a role in the generation of potentially immunostimulatory forms of autoantigens. In the present study, we examined changes in the Golgi complex and associated autoantigens during apoptosis and necrosis. Immunofluorescence analysis showed that the Golgi complex was altered and developed distinctive characteristics during apoptosis and necrosis. In addition, immunoblotting analysis showed the generation of antigenic fragments of each Golgi autoantigen, suggesting that they may play a role in sustaining autoantibody production. Further studies are needed to determine whether the differences observed in the Golgi complex during apoptosis or necrosis may account for the production of anti-Golgi complex autoantibodies.     

The specificity of human anti-cytochrome c autoantibodies that arise in autoimmune disease.

Cytochromes c (cyt c) are among the best characterized model Ag because their amino acid sequences and tertiary structures are well defined. One unique aspect of cyt c as an immunogen is its ability to induce autoantibody responses in animal models, although no pathology resulting from these responses has been reported. In this study, the presence and specificity of autoantibodies to cyt c were investigated in patients with SLE and related connective tissue diseases. Anti-cyt c antibodies were found in approximately 7% of patient sera and were statistically associated with the expression of antimitochondrial antibodies but were not statistically associated with any disease subset among those represented. Anti-cyt c was not associated with the presence of autoantibodies to DNA, histones, Ro, La, or Sm autoantigens. Most of the autoantibodies were specific for native or native-like forms of cyt c but antibodies to denatured forms were also apparent. Autoantibody binding was shown to be directed predominantly at selected sites of evolutionary variability within cyt c. The specificity of the human anti-cyt c autoantibodies appear to be similar to that of mouse anti-human cyt c antibodies and to autoantibodies elicited in mice against rat (mouse) cyt c.     

Class-specific binding of two aminoacyl-tRNA synthetases to annexin, a Ca2+- and phospholipid-binding protein

Annexins are a family of Ca2+/phospholipid-binding proteins that have diverse functions. To understand the function of annexin in Physarum polycephalum, we searched for its binding proteins. Here we demonstrate the presence of two novel annexin-binding proteins. The homology search of partial amino acid sequences of these two proteins identified them as aminoacyl-tRNA synthetases (ARSs). Furthermore, antibody against aminoacyl-tRNA synthetases cross-reacted with one of two proteins. Our results imply the interaction between intracellular membrane dynamics and protein translation system, and may give a clue to understand the mechanism of some myositis diseases, which have been known to produce autoantibodies against ARSs.     

Pediatric autoimmune liver diseases: the molecular basis of humoral and cellular immunity

Pediatric autoimmune liver disease is mainly represented by two similar liver disorders: autoimmune hepatitis (AIH) and autoimmune sclerosing cholangitis (ASC), both characterized by hypergammalobulinemia, interface hepatitis and the presence of a wide range of circulating autoantibodies. Although similar features are seen in AIH and inflammatory bowel disease, histological biliary changes are more common in ASC. In addition to their role as diagnostic markers, autoantibodies, such as anti-extractable nuclear antigen (ENA) antibodies and liver kidney microsomal antibody type 1 (LKM1) may be involved directly in inducing aggressive liver diseases. Although the cellular immune response in pediatric autoimmune liver disease has been less intensively investigated than humoral immunity, the importance of antigen specific T cells has been explored. Both alphabeta and gammadelta T cells derived from either peripheral blood and liver biopsies have highly heterogeneous TCR gene usage and cytolytic activity has been demonstrated. There have been attempts to seek triggers of liver autoimmunity and several sequences shared in common between autoantigens and hepatotropic viruses, namely hepatitis B, C and cytomegalovirus have been identified. The presence of cross-reactivity between homologous sequences, especially between HCV and cytochromes, supports the possibility that molecular mimicry plays a role in the induction of autoantibodies and autoreactive cytotoxic T cells.     

Nonhistone nuclear antigens reactive with autoantibodies. Immunofluorescence studies on distribution in synchronized cells

Sera from patients with certain autoimmune diseases tht contained autoantibodies to nonhistone nuclear antigens were used as reagents in an indirect immunofluorescent study. The distribution of these nuclear antigens was determined in synchronized human B lymphoid cells. Autoantibodies to Sm antigen, nuclear ribonucleoprotein complex and SS- B antigen were used. Although all three nonhistone antigens appeared to show speckled nuclear straining patterns in the Go phase, different patterns of staining were present at other periods of the cell cycle. The SS-B antigen showed a distinctly nucleolar localization during the G1/early S phase. These studies demonstrate that autoantibodies occurring in certain human diseases can be useful reagents for the immunohistological localization of nuclear macromolecules and for tracing their pathways during different phases of cell growth and differentiation.     

Recurrent staphylococcal cellulitis and subcutaneous abscesses in a child with autoantibodies against IL-6

We investigated an otherwise healthy patient presenting two episodes of staphylococcal cellulitis and abscesses, accompanied by high fever and biological signs of inflammation but, paradoxically, with no detectable increase in serum levels of C-reactive protein (CRP), an IL-6-responsive protein synthesized in the liver. Following in vitro activation of whole blood cells from the patient with multiple cytokines, TLR agonists, heat-killed bacteria, and mitogens, we observed a profound and specific impairment of IL-6 secretion. However, the patient's PBMCs, activated in the same conditions but in the absence of the patient's plasma, secreted IL-6 normally. The patient's serum contained high titers of IgG1 autoantibodies against IL-6, which specifically neutralized IL-6 production by control PBMCs as well as IL-6 responses in the human hepatocellular carcinoma cell line Hep3B. These anti-IL-6 autoantibodies were detected over a period of 4 years, in the absence of any other autoantibodies. Our results indicate that these Abs probably prevented an increase in CRP concentration during infection and that impaired IL-6-mediated immunity may have contributed to staphylococcal disease. Patients with severe bacterial infections and low serum CRP concentrations should be tested for anti-IL-6 autoantibodies, especially in the presence of other clinical and biological signs of inflammation.     

RNA recognition by autoantigens and autoantibodies

The La, Ro, Sm and RNP autoantigens have been intensely studied over the past decade since cDNAs encoding autoantigens have become available. Most of these autoantigens are closely associated with RNA in RNP particles and molecular studies have provided insights into their modes of recognition and binding to RNA. For example, a common RNA Recognition Motif (RRM) was found to be a critical component of the RNA-binding domain of these autoantigens and the three dimensional structure of the RRM has been solved. As described in other articles in this series, the presence of La, Ro, Sm and RNP autoantibodies correlates with disease subsets, such as Sjogren's syndrome, systemic lupus erythematosus and other connective tissue diseases. Immunological analysis of sera from autoimmune patients using recombinant autoantigens has revealed that multiple epitopes reside along the proteins and these represent both continuous and discontinuous (conformational) autotopes. Findings to date support a model of autoantibody induction which involves the direct presentation of proteinaceous autoantigens to the immune system. Circumstantial evidence has suggested that immunological crossreactivity between systemic autoantigens and structural components of infectious agents may play an initial role in the autoimmune response to certain antigens. However, the etiology of autoimmune diseases is probably multifactoral with genetic and other immune features acting on the organismal level. In addition, RNA molecules themselves can be autoantigens with higher order structural conformations which are recognized by RNP-type autoantibodies. Immune crossreactivity and/or direct presentation may generate autoantibodies reactive with conformational RNA epitopes. If crossreactivity with components of cellular or infectious agents give rise to RNA epitopes, they may represent structural or functional mimetics of the primary epitopes that actually drive the response. These ideas are discussed with respect to the role of mimetic processes in molecular recognition during autoimmunity.     

Anti-cytokine autoantibodies: Epiphenomenon or critical modulators of cytokine action

Low amounts of high-affinity autoantibodies to various cytokines have been detected in sera from healthy donors. Their levels, although highly variable, are increased in the circulation of patients subjected to cytokine therapy or suffering from a variety of immunoinflammatory diseases. It has been suggested that these autoantibodies play a regulatory role in the intensity and duration of an immune response. The antibodies may prevent the binding of a cytokine to its specific cell surface receptor thereby neutralizing its biological activityin vivo. They may also act as carrier proteins preventing the rapid elimination of a cytokine from the circulation and thus increase its bioactivity. Additionally or alternatively, autoantibodies may modulate cytokine-induced intracellular signal transduction pathways or trigger complement-mediated cytotoxicity towards cells carrying membrane-bound cytokines. The autoantibodies may exert their regulatory role in compliance with other factors that control cytokine activity, including soluble cytokine receptors, cell surface decoy receptors, and receptor antagonists. Although not favored by many investigators, a less sophisticated role for naturally occurring anti-cytokine autoantibodies should be considered as well. Recent evidence has shown that autoantibodies are generated at a high frequency as part of a response to foreign antigens. These antibodies are produced by B cells arising from the process of somatic mutation. Thus anti-cytokine autoantibodies may be the result of a “leaky” B cell response triggered by immunoinflammatory processes. High-titered autoantibodies induced by cytokine therapy are of clinical concern since their occurrence is often associated with the loss of response to treatment. Moreover, they may also neutralize endogenously produced cytokines with possible pathological consequences. In this paper we have reviewed the available information on the biological and clinical significance of both naturally occurring and therapeutically induced anti-cytokine autoantibodies in animals and man with the emphasis on antibodies directed to interferons.     

Characterization and potential clinical applications of autoantibodies against cytokines

Autoantibodies recognizing cytokines arise in certain patients during the course of therapy with recombinant cytokines, although they may arise spontaneously as well. They are typically high avidity and in vitro neutralizing IgG antibodies present in picomolar to nanomolar concentrations. Methodology is therefore critical. Quantitative studies based on sound methodology strongly indicate that nanomolar levels of cytokine autoantibodies are likely to be involved in a number of “new” syndromes such as acquired immune deficiencies, lung diseases, and certain age-related manifestations. There are many ways in which the autoantibodies could be naturally induced, and they have been experimentally induced with ease. Therefore, a new therapeutic concept of inducing cytokine autoantibodies via anti-cytokine vaccination is currently rapidly emerging.     

Recurrent utilization of genetic elements in V regions of antinucleic acid antibodies from autoimmune mice.

Two IgG anti-DNA and two IgG anti-RNA autoantibodies derived from lupus prone NZB/NZW F1 mice have been analyzed for their Ag fine specificities and for their H and L chain V-region sequences. A remarkable similarity of VH gene sequences with previously sequenced antinucleic acid autoantibodies (Eilat, D., D. M. Webster and A. R. Rees. J. Immunol. 141:1745, 1988) was noted. This finding indicates that a small number of unique VH genes is involved in this autoimmune response and that the sequences of these genes are correlated with the different specificities for the autoantigen. The VK sequences appeared, by computer search, to be selected nonrandomly, but their use was not restricted to autoantibodies. An additional striking feature was evident in the construction of the D region elements, giving rise to CDR3 peptides that can interact with DNA and RNA. These constructs probably include D-D fusion products, which are relatively rare in Ig rearrangements.     

Recovery of ovulatory menstrual cycles under hydrocortisone in two amenorrheic women with isolated corticotropin deficiency

Two amenorrheic women presenting clinical signs of adrenal insufficiency were shown to have isolated corticotropin deficiency (ICD). LH and FSH were normally responsive to GnRH. The occurrence of this disease during the postpartum and the presence of autoantibodies against corticotropic cells in one case may indicate that ICD was a sequela of an autoimmune hypophysitis. The presence of amenorrhea, while the gonadotroph was not damaged, and the reappearance of ovulatory menstrual cycles under the sole effect of hydrocortisone replacement therapy suggest that cortisol deficiency may by itself alter the gonadal function.     

Mapping of the herpes simplex virus DNA sequences present in herpes simplex virus type-1 thymidine kinase-transformed cells

Analyses of the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase-transformed (HSVtk+) mouse cell lines have revealed that these cells contain relatively large and variable portions of the viral genome. Two of these cell lines do not contain the viral DNA sequences known to encode the early viral genes normally responsible for regulating tk gene expression during lytic HSV infections. This finding suggests that cell-associated viral tk gene expression may be regulated by cellular rather than viral control mechanisms. In addition, we have compared the viral DNA sequences present in one unstable HSVtk+ cell line to those present in tk- revertant and tk+ rerevertant cell lines sequentially derived from it. Our results have shown that within the limits of sensitivity of our mapping approach, these three related cell lines contain the same set of viral DNA sequences. Thus, gross changes in viral DNA content do not appear to be responsible for the different tk phenotypes of these cells.     

Poliovirus neutralization by antibodies to internal epitopes of VP4 and VP1 results from reversible exposure of these sequences at physiological temperature.

Antisera were raised against peptide sequences that are normally internal in the poliovirus virion. These antisera contain neutralizing activity, but this neutralizing activity is dependent on coincubation of the virus and antisera at 37 degrees C. Immunoprecipitation analyses demonstrate that the neutralization is due to exposure of these normally internal sequences at 37 degrees C and subsequent antibody binding. Exposure of these sequences is reversible. These data demonstrate that the poliovirus particle is a dynamic entity that is capable of undergoing conformational alterations at physiological temperatures. This conformational flexibility provides an explanation for earlier observations of virus neutralization by antibodies to internal epitopes which can be accommodated within the framework of existing models for antibody-mediated neutralization of viral infectivity. Analogies between the sequences which are reversibly exposed at 37 degrees C with those which are irreversibly exposed upon receptor binding suggest that the observed conformational dynamics also may play a role in cell entry.