Mitochondrial Antigens Research Articles

Synthesis of interleukin-1β in primary biliary cirrhosis: Relationship to treatment with methotrexate or colchicine and disease progression

Primary biliary cirrhosis (PBC) is a chronic, progressive, cholestatic liver disease. Interleukin-1β (IL-1β) may play a role in the pathogenesis of PBC by contributing to altered immune function and fibrosis. Colchicine or methotrexate has some beneficial effects in the treatment of PBC, and also affects interleukin-1 (IL-1β). Therefore, we prospectively studied the synthesis of IL-1β by peripheral blood mononuclear cells (PBMC) from 42 patients with PBC entered into a randomized, double-blind, double-dummy controlled trial of colchicine and methotrexate. PBMC obtained at entry, 6, 12, 18, and 24 months were stimulated to produce IL-1β with phytohemagglutinin (PHA), lipopolysaccharide (LPS), Staphylococcus epidermidis, recombinant IL-2, or mitochondrial antigen. Patients in the two treatment groups did not differ at entry in biochemical measures or liver histological stage. Over 24 months in both groups, serum bilirubin and histologic stage remained stable and alkaline phosphatase decreased significantly. For all patients, synthesis of IL-1β increased constitutively and in response to immune-mediated stimulants (PHA, IL-2, and mitochondrial antigen) but not the bacterial stimulants LPS or S epidermidis. Compared with levels of IL-1β at entry, PHA induced increases for patients treated with methotrexate (12, 18, and 24 months) or colchicine (18 and 24 months). At 24 months, IL-2-induced IL-1β synthesis was increased in patients treated with methotrexate, whereas S epidermidis-induced IL-1β was enhanced in colchicine-treated patients. Before treatment, IL-1β production did not relate to severity of disease except in response to S epidermidis. Notably, in patients with stable or improving liver histological stage, IL-1β synthesis increased dramatically at 12 months in response to IL-2 and S epidermidis, and also to LPS by 24 months. Alterations in IL-1β synthesis in patients with PBC may reflect underlying immunoregulatory changes that contribute to disease progression or stabilization.

Synthesis of interleukin-1β in primary biliary cirrhosis: Relationship to treatment with methotrexate or colchicine and disease progression

Primary biliary cirrhosis (PBC) is a chronic, progressive, cholestatic liver disease. Interleukin-1β (IL-1β) may play a role in the pathogenesis of PBC by contributing to altered immune function and fibrosis. Colchicine or methotrexate has some beneficial effects in the treatment of PBC, and also affects interleukin-1 (IL-1). Therefore, we prospectively studied the synthesis of IL-1β by peripheral blood mononuclear cells (PBMC) from 42 patients with PBC entered into a randomized, doubleblind, double-dummy controlled trial of colchicine and methotrexate. PBMC obtained at entry, 6, 12, 18, and 24 months were stimulated to produce IL-1β with phytohemagglutinin (PHA), lipopolysaccharide (LPS), Staphylococcus epidermidis, recombinant IL-2, or mitochondrial antigen. Patients in the two treatment groups did not differ at entry in biochemical measures or liver histological stage. Over 24 months in both groups, serum bilirubin and histologic stage remained stable and alkaline phosphatase decreased significantly. For all patients, synthesis of IL-1β increased constitutively and in response to immune-mediated stimulants (PHA, IL-2, and mitochondrial antigen) but not the bacterial stimulants LPS or S epidermidis. Compared with levels of IL-1β at entry, PHA induced increases for patients treated with methotrexate (12, 18, and 24 months) or colchicine (18 and 24 months). At 24 months, IL-2-induced IL-1β synthesis was increased in patients treated with methotrexate, whereas S epidermidis-induced IL-1β was enhanced in colchicine-treated patients. Before treatment, IL-1β production did not relate to severity of disease except in response to S epidermidis. Notably, in patients with stable or improving liver histological stage, IL-1β synthesis increased dramatically at 12 months in response to IL-2 and S epidermidis, and also to LPS by 24 months. Alterations in IL-1β synthesis in patients with PBC may reflect underlying immunoregulatory changes that contribute to disease progression or stabilization. (Hepatology 1995;22:518-524.)

Mitochondrial antigens, molecular mimicry and autoimmune disease

The immune system is normally tolerant to mitochondrial self-antigens, but responsive against bacteria. Low-titre anti-mitochondrial antibodies (AMA) might be involved in this discrimination. Tolerance is broken in diseases characterised by high titre AMA. Some of these AMA, against cardiolipin, cross-react with DNA. The best studied AMA are those characterising primary biliary cirrhosis (PBC). These are directed against E2 subunits of the oxo-acid dehydrogenase complexes, and also against subunits E1 α, E1 β and X of the pyruvate dehydrogenase complex. AMA of PBC patients also react with bacterial E2s. Reactivities are primarily peptide-specific but with cross-reactivity between mitochondrial and microbial antigens and between E2s of respective complexes. Immunodominant epitopes, for anti E2 AMA, include the conserved sequence flanking the site of lipoyl attachment. It is proposed that the initial stimulus for antibody production is chronic urinary tract infection. AMA themselves are not pathogenic, but CD4 + T-cells would be primed, recognising the lipoyl domain epitope in association with class II HLA. Inappropriate expression of class II antigens on bile duct epithelia, (as found in PBC), might lead to presentation of a particular fragment of HLA-DR α, known to be a major MHC presented self-peptide in the mouse. That sequence strongly mimics the lipoyl domain and might be recognised by primed T-cells, initiating the autoimmune cascade. In the mouse, a peptide of ND1 of Complex I is presented in association with class I MHC. Cells exhibiting somatic mutation of such a peptide might thus be subject to attack by CD8 + T-cells. If such peptides were presented by class II HLA, autoimmune diseases might arise, related to mimicry between such peptides and microbial sequences and/or self-antigens. These considerations might apply in Leber's disease and in age-related pathology.

Mitochondrial constituents of corpora amylacea and autofluorescent astrocytic inclusions in senescent human brain

Corpora amylacea (CA) are cytoplasmic inclusions that accumulate in human brain in the course of normal aging, and to an even greater extent, in Alzheimer's disease and other neurodegenerative conditions. In senescent and Alzheimer-diseased human brains, astrocytes in limbic and periventricular regions exhibit red autofluorescent inclusions, homologous to Gomori-positive astrocyte granules previously described in the brains of aging rodents and other vertebrates. We have shown that Gomori inclusions in situ and in culture are derived from autophagocytosed mitochondria exhibiting iron-mediated peroxidase activity. In the human brain, the autofluorescent inclusions share many properties with CA. Both types of inclusion progressively accumulate in periventricular regions with advancing age, are largely astrocytic in origin, and contain various heat shock proteins and ubiquitin. Using histochemistry in conjunction with cofocal microscopy, we demonstrated that both CA and the red autofluorescent granules exhibit non-enzymatic peroxidase activity and an affinity for CAH and PAS. The only major divergent histochemical feature between the Gomori-positive astrocyte granules and CA is the presence of orange-red autofluorescence in the former and the absence of endogenous fluorescence in the latter. On the basis of numerous shared topographic and histochemical features, we hypothesized that CA are largely derived from autofluorescent (Gomori-positive) astrocyte granules which reside in periventricular regions of the senescent CNS. Immunofluorescent labeling and laser scanning confocal microscopy demonstrated consistent colocalization of the mitochondrial proteins, sulfite oxidase, and heat shock protein 60, to both CA and the autofluorescent astroglial inclusions. In addition, both CA and the autofluorescent astrocyte granules exhibit staining for DNA which colocalizes to mitochondrial antigens and therefore likely represents mitochondrial nucleic acid in dual-labeled preparations. These observations suggest that a) Gomori-positive astrocyte granules in human brain are homologous to those described in rodents, b) Gomori-positive granules may be structural precursors of CA in senescent human brain, and c) in the aging human brain, degenerate mitochondria within periventricular astrocytes give rise to autofluorescent cytoplasmic granules and corpora amylacea.

Establishment and structural analysis of human mAb to the E2 component of the 2-oxoglutarate dehydrogenase complex generated from a patient with primary biliary cirrhosis.

We established one Epstein-Barr virus-transformed B cell hybrid clone producing human mAb of the IgG class to the 2-oxoglutarate dehydrogenase complex (OGDC) for the first time from the peripheral B lymphocytes of a patient with primary biliary cirrhosis (PBC). This mAb, designated mAbM37GO37, specifically bound to OGDC and its dissociation constant with OGDC was calculated to be 3.70 x 10(-10) mol/l. mAbM37GO37 stained murine stomach/kidney cryostat sections in a typical immunofluorescence pattern of antimitochondrial antibody (AMA). Western blotting analysis revealed that mAbM37GO37 reacted with an E2 component of OGDC but not with other components of OGDC nor pyruvate dehydrogenase complex (PDC). Furthermore, mAbM37GO37 completely inhibited the enzymatic activity of OGDC. In order to determine the structure and genetic origin of anti-OGDC autoantibody, we cloned and sequenced the Ig heavy and light chain variable regions of mAbM37GO37. This mAb used the VHIII family member, V3-7, and the V kappa IV family member. The amino acid difference between the expressed V genes of this mAb and respective putative germline genes was concentrated within the complementarity determining regions (CDR) rather than the framework regions (FR). The R:S mutation ratio was high in the CDR and low in the FR. These features suggested that the immune response to OGDC is similar to that to exogenous antigen, and that the heavy and light chain variable regions of the anti-OGDC antibody undergo somatic hypermutation through antigen-driven clonal selection. This human mAb to OGDC, which was established for the first time from a patient with PBC and characterized at the molecular level, would be a valuable tool to study the B cell autoepitopes of OGDC, to clone as yet undetermined full length cDNA encoding OGDC and to dissect the autoimmune response to mitochondrial antigens in PBC.

The association of primary biliary cirrhosis and systemic sclerosis is not accounted for by cross reactivity between mitochondrial and centromere antigens.

A proportion of patients with primary biliary cirrhosis (PBC) develop the CREST variant of systemic sclerosis (SSc) and in these individuals antimitochondrial antibodies (AMA) and anticentromere antibodies (ACA) coexist. Immunological cross-reactivity between mitochondrial and centromere-associated antigens might account for the clinical and serological overlap between these conditions. Therefore, antibodies were affinity purified from the 70 kD polypeptide corresponding to the E2 component of the pyruvate dehydrogenase complex (PDHC) and from CENP-C, a 140 kD centromere-associated protein, to examine this possibility. Although the purified antibodies reacted with their corresponding antigens, no evidence of shared determinants between the 70 kD protein and CENP-C could be detected whether the antibodies were prepared from monospecific sera or from sera containing both AMA and ACA. Therefore, AMA and ACA present discrete autoantibody populations which may coexist in the same patient and may influence the clinical picture but have both structural and immunologically independent antigenic targets.

Cross-reactivity of anti-Mycobacterium gordonae antibodies with the major mitochondrial autoantigens in primary biliary cirrhosis

Primary biliary cirrhosis is a chronic cholestatic liver disease associated with autoimmune disorders. Antimitochondrial autoantibodies and granulomatous portal lesions are characteristic in primary biliary cirrhosis. Since granuloma may be induced by Mycobacteria, and there is evidence implicating Mycobacteria as infectious agents capable of initiating autoimmunity, a study was performed to determine the presence of antibodies against 10 atypical Mycobacteria in 19 patients with primary biliary cirrhosis, and in 35 controls (25 patients with other chronic liver diseases and 10 healthy subjects). All primary biliary cirrhosis sera and none of the controls reacted with the extract from Mycobacterium gordonae, showing identical recognition profiles with two polypeptides of 70-65 and 55 kDa. No other reaction was found in primary biliary cirrhosis patients and in controls with the extracts from the other nine atypical Mycobacteria tested. Eluted immunoglobulins which reacted with the 70-65 and 55 kDa polypeptides from M. gordonae, bound to the mitochondrial antigens PDH-E2 and BCKDH-E2. Furthermore, when the extract from M. gordonae was tested with eluted immunoglobulins from recognized PDH-E2 and BCKDH-E2 by primary biliary cirrhosis patients, we observed both 70-65 and 55 kDa polypeptides. These data indicate that antibodies to M. gordonae, found in all primary biliary cirrhosis patients, cross-react with the major mitochondrial targets of the disease. We suggest that M. gordonae may play a potential pathogenic role in primary biliary cirrhosis.

Characterization and clinical relevance of liver-pancreas antibodies in autoimmune hepatitis

The isolation of a marker antigen from rat liver and pancreas tissue, which reacts with antibodies in a subtype of autoimmune hepatitis by complement fixation test, enzyme-linked immunosorbent assay and Western blot, is described. The liver-pancreas antigen could be detected in tissue from different human or animal organs, but liver and pancreas yielded the highest activity. A highly specific antigen fraction was obtained by gel filtration and ion exchange chromatography with a 100,000 g supernatant from rat liver tissue, and this preparation was shown to be devoid of nuclear, mitochondrial and microsomal antigens and cytokeratin 8 and 18, as demonstrated by appropriate marker antibodies. These data and absorption studies with cell organelles indicate that liver-pancreas antigen is a cytosolic protein. By Western blotting, two major epitopes at molecular weights 52 kD and 48 kD could be visualized. Sera from 175 patients previously shown to have high complement-fixing activity to a nonpurified liver-pancreas antigen fraction were further analyzed. All were positive by enzyme-linked immunosorbent assay with the purified liver-pancreas antigen fractions, and 111 were also positive by Western blot. Eighty-six sera reacted with the 52-kD determinant, 33 with the 48-kD determinant and 2 with both determinants. In 117 of the 175 patients, antibody to liver-pancreas antigen was associated with other autoantibodies known to characterize subgroups of autoimmune hepatitis. Thus 19 patients had antibodies to nuclei and 96 to actin but none to liver-kidney microsomes, hereby suggesting that antibody to liver-pancreas antigen may define another subgroup of autoimmune hepatitis.(ABSTRACT TRUNCATED AT 250 WORDS)

M4 and M9 Antibodies in the Overlap Syndrome of Primary Biliary Cirrhosis and Chronic Active Hepatitis: Epitopes or Epiphenomena?

Before the identification of the major mitochondrial antigens of primary biliary cirrhosis as components of the 2-oxo-acid dehydrogenase enzyme family, mitochondrial autoantigens were believed to be extremely heterogeneous and were divided into nine subtypes termed M1 to M9. This classification was based on the data derived from the relatively nonspecific biochemical and immunological techniques that were available. After the cloning and definition of the major autoantigens, more than 95% of the sera of patients with primary biliary cirrhosis were found to react with components of the 2-oxo-dehydrogenase enzymes; these enzymes correspond to the old M2 classification. Two other "M" species, dubbed M4 and M9, have attracted significant attention because they have been postulated to be prognostic indicators and more recently have been tentatively identified respectively as sulfite oxidase (EC 1.8.3.1) and glycogen phosphorylase (EC 2.4.1.1). Indeed, patients with the "overlap syndrome" are reported to have antibodies to M4 and a poor prognosis, whereas patients with antibodies to M9 have a favorable prognosis. To address the significance and definition of M4 and M9, we performed in-depth studies of sera from 11 patients with the overlap syndrome, 75 patients with primary biliary cirrhosis, 19 chronic active hepatitis patients, 13 patients with primary sclerosing cholangitis, 10 patients with cholangiocarcinoma, 20 patients with systemic lupus erythematosus, 20 patients with alcoholic cirrhosis, 17 patients with scleroderma and 30 normal individuals, using techniques of ELISA, complement fixation, immunoblotting and enzyme inhibition. We report herein that we were unable to show any disease-specific reactivity toward the proposed M4 and M9 antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

M4 and M9 antibodies in the overlap syndrome of primary biliary cirrhosis and chronic active hepatitis: Epitopes or epiphenomena?

Before the identification of the major mitochondrial antigens of primary biliary cirrhosis as components of the 2-oxo-acid dehydrogenase enzyme family, mitochondrial autoantigens were believed to be extremely heterogeneous and were divided into nine subtypes termed M1 to M9. This classification was based on the data derived from the relatively nonspecific biochemical and immunological techniques that were available. After the cloning and definition of the major autoantigens, more than 95% of the sera of patients with primary biliary cirrhosis were found to react with components of the 2-oxo-dehydrogenase enzymes; these enzymes correspond to the old M2 classification. Two other “M” species, dubbed M4 and M9, have attracted significant attention because they have been postulated to be prognostic indicators and more recently have been tentatively identified respectively as sulfite oxidase (EC 1.8.3.1) and glycogen phosphorylase (EC 2.4.1.1). Indeed, patients with the “overlap syndrome” are reported to have antibodies to M4 and a poor prognosis, whereas patients with antibodies to M9 have a favorable prognosis. To address the significance and definition of M4 and M9, we performed in-depth studies of sera from 11 patients with the overlap syndrome, 75 patients with primary biliary cirrhosis, 19 chronic active hepatitis patients, 13 patients with primary sclerosing cholangitis, 10 patients with cholangiocarcinoma, 20 patients with systemic lupus erythematosus, 20 patients with alcoholic cirrhosis, 17 patients with scleroderma and 30 normal individuals, using techniques of ELISA, complement fixation, immunoblotting and enzyme inhibition. We report herein that we were unable to show any disease-specific reactivity toward the proposed M4 and M9 antigens. We offer potential explanations for false-positive readings and, most importantly, propose that the “M” nomenclature be discontinued and that autoantigens be designated by their proven and reproducible biochemical identity. (HEPATOLOGY 1992;16:1128–1136.)

Use of designer recombinant mitochondrial antigens in the diagnosis of primary biliary cirrhosis.

The appearance of autoantibodies against mitochondria in patients with primary biliary cirrhosis has been known for more than 25 yr. In the past, based on the biochemical complexity of the mitochondrion and the use of crude extracts for immunodiagnosis, a degree of nonspecificity in assaying for antibodies to mitochondria has been present. This problem has been largely circumvented by the cloning of the mitochondrial antigens and the identification of the E2 subunits of the pyruvate dehydrogenase complex and the branched chain 2-oxo-acid dehydrogenase complex as the major and immunodominant autoantigens of primary biliary cirrhosis. More than 90% of patients with primary biliary cirrhosis have been shown to react with one or both of these enzymes using either recombinant antigen or purified native protein. Approximately 10% of patients recognize only E2 subunits of branched chain 2-oxo-acid dehydrogenase complex and not pyruvate dehydrogenase complex. Such patients would be missed by diagnostic assay that has a low sensitivity to antibodies against E2 subunits of branched chain 2-oxo-acid dehydrogenase complex. The use of recombinant and biochemically pure antigens has permitted structural and conformational analysis of epitope mapping. We have taken advantage of the antigenic mapping studies of both primary biliary cirrhosis and branched chain 2-oxo-acid dehydrogenase complex E2 subunits and designed a molecule that expresses the immunodominant epitopes of both. Using this dual-headed molecule that coexpresses the epitope of two different antigens, we report herein a sensitive and reproducible assay for antibodies to mitochondria in patients with primary biliary cirrhosis.(ABSTRACT TRUNCATED AT 250 WORDS)

A flow cytometric method to detect anti-pyruvate dehydrogenase antibody in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by the presence of anti-mitochondrial antibodies specifically directed against the M2 group of mitochondrial antigens. Recently, the E-1, the E-2, and protein X components of pyruvate dehydrogenase enzyme complex have been identified as the major antigens within the M2 group of autoantigens. An immunoassay using pyruvate dehydrogenase enzyme complex as a specific antigen for the diagnosis of PBC was developed. Pyruvate dehydrogenase enzyme complex was attached to polystyrene microbeads, incubated with sera from PBC patients (n = 18), normal controls (n = 50), or patients with other autoimmune diseases (n = 26), followed by incubation with a second fluorescein isothiocyanate conjugated goat anti-human immunoglobulin and then analyzed by flow cytometry. High numbers of fluorescence channels (mean, 1,693 +/- 846) were obtained for all PBC sera except for two patients. Compared to the conventional anti-mitochondrial antibody assay, the assay had a sensitivity rate of 94% and a specificity rate of 100%. The reactive antibodies are predominantly of the immunoglobulin G3 subclass. Their levels could be correlated with the histopathologic stages of PBC. These results were corroborated by immunoblotting. Sera from patients with later stages of PBC strongly reacted with pyruvate dehydrogenase enzyme complex components, E1 alpha, and protein X.

Levels of autoantibodies against beta-adrenergic receptors and mitochondrial antigens in sera of cardiomyopathic patients estimated by ELISA method.

Levels of autoantibodies against beta-adrenergic receptors and mitochondrial antigens were estimated by the ELISA method in a group of 41 cardiomyopathic children. These results were compared with results obtained from 26 sera of a control group. In the group of cardiomyopathic patients 11 sera with significantly increased levels of autoantibodies against beta-adrenergic receptors and seven sera with increased levels of autoantibodies against mitochondrial antigens were found. In no sample of the control group did we observe sera with increased levels of autoantibodies. Statistical significance of this data was evaluated by chi 2-test (p less than 0.005 or p less than 0.05, resp.).

The relationship between in vitro fertilization and naturally occurring antibodies: evidence for increased production of antiphospholipid autoantibodies

Assessment of possible effects of ovarian stimulation during in vitro fertilization (IVF) treatment cycles on circulating levels of antiphospholipid and antinuclear autoantibodies. The study was performed prospectively. Sera were obtained at three time points along IVF treatment cycle. Levels of autoantibodies directed against nuclear components, mitochondrial antigens, and phospholipids were determined using enzyme-linked immunosorbent assay. Thirty-five patients, who underwent at least one previous IVF attempt, and 36 age- and sex-matched controls were analyzed. All participants were randomly selected. The mean levels of antiphospholipid (but not antinuclear) autoantibodies in sera from IVF-treated patients were found to be significantly higher than the corresponding values of the control group (for immunoglobulin [Ig]M isotype: anticardiolipin, antiphosphatidyl L-serine; for IgG isotype: anticardiolipin, antiphosphatidyl L-serine, and antiphosphatidylcholine; P less than 0.0001, assessed by Mann-Whitney test). The autoantibody levels remained more or less constant at different time points along the treatment cycle. No correlation with age and number of previous IVF cycles was demonstrated. Serum levels of antiphospholipid (but not antinuclear) autoantibodies increase after IVF treatment. Based on these preliminary data, it is not yet possible to estimate if the observed changes in autoantibody levels might have any future clinical influence on infertile patients undergoing IVF treatment.

An automated microassay for enzyme inhibitory effects of M2 antibodies in primary biliary cirrhosis

In primary biliary cirrhosis (PBC), autoantibodies are produced to the M2 group of mitochondrial antigens, of which a major constituent is the E2 subunit of the pyruvate dehydrogenase complex (PDC). These antibodies, in addition to conventional reactivities with PDC, characteristically inhibit the catalytic function of PDC in vitro, as judged by a macroinhibition assay based on spectrophotometry. We describe a microinhibition assay adapted for microtitre plates and for an automated readout of results by an ELISA plate reader. We show that this microassay has a sensitivity similar to that of the macroassay. In a study of 83 sera, from PBC and other diseases, the inhibitory microassay proved specific for PBC. This automated inhibitory microassay for PBC-sera could become a primary laboratory procedure for the diagnosis of PBC, particularly because it may identify antibodies to the actual autoepitope on the PDC-E2.

Mitochondrial antigens and antibodies in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is considered to be an autoimmune chronic liver disease. It is characterized by progressive inflammatory destruction of intrahepatic bile ducts which results in cholestasis and which may progress to cirrhosis and liver failure. It is a disease which occurs in females in 90% of cases. The diagnosis is most commonly made in middle aged women who present with itching and usually with jaundice. However, less severe cases are being diagnosed because ofcholestatic liver function tests or the presence of antimitochondrial antibodies (AMAs) in the serum. The latter are the most characteristic feature of PBC.1 These AMAs are non-organand nonspecies-specific and are of both the IgG and IgM classes. They were first detected in PBC patients in 1965 by Walker et al.2 by indirect immunofluorescence tests on cryostat sections. To date, 9 different AMA types (anti-M I to anti-M9) have been defined, using immunofluorescence and complement fixation tests (CFTs), enzyme linked immunosorbent assay (ELISA) and Western blotting.35 Four of these AMAs, anti-M2, -M4, -M8 and -M9 are associated with PBC. The others anti-MI, -M3, -M5, -M6 and -M7 are mainly associated with non-hepatic disorders.6 The past 4 years have seen a great expansion in publications related to the immunology ofPBC due to the identification ofthe major M2 antigens and the use of molecular biology techniques. These give good prospects for a better understanding of the pathogenesis of the disease.

Generation of T cells with lytic specificity for atypical antigens. I. A mitochondrial antigen in the rat.

F1 rats primed with normal parental strain lymphocyte populations and restimulated in culture with parental lymphoblasts generate potent cytotoxic T cell responses to unusual antigen systems. Here we describe in the Lewis (L)/DA anti-DA combination an antigen system most likely of mitochondrial origin with the following properties: it is transmitted maternally from DA strain females, inherited in an extra-chromosomal manner, restricted by class I RT1Aa major histocompatibility complex gene products, extinguished on target cells treated with chloramphenicol, and its pattern of expression in different rat strains correlates with restriction fragment-length polymorphisms of mitochondrial DNA. Sequence analysis of the rat ND1 gene indicates that the maternally transferred factor in the rat is not a homologue of the maternally transmitted factor responsible for the mitochondrial antigen in mice. In keeping with its inheritance from DA females, this antigen is present on target cells from (DA female x L male)F1 donors and all other F1 combinations derived from DA female parents, but absent from target cells from some F1 combinations (L/DA and Wistar-Furth [WF]/DA) derived from DA strain males. The presence of this antigen in other F1 combinations (Brown Norway [BN]/DA, August 2880 [AUG]/DA, and PVG/DA) indicates that this mitochondrial antigen system is shared by the DA, BN, and PVG strains, but not by the L and WF strains.

Generation of T cells with lytic specificity for atypical antigens. III. Priming F1 animals with antigen-bearing cells also having reactivity for host alloantigens allows for potent lytic T cell responses.

Here, we explore the conditions required for generating two different highly potent F1 antiparental killer cell populations to unusual antigens in rats. The first, L/DA anti-DA, has lytic specificity for two antigen systems: MTA, a mitochondrial antigen expressed on DA and DA Lewis (L) target cells restricted by RT1A class I molecules; and H, an antigen that maps to the class I-like RT1C region and is present only on parental target cells from donors homozygous at the major histocompatibility complex. The second killer population is generated in the reciprocal DA/L anti-DA combination and has lytic specificity only for the H antigen system. We show that the killer cells are T cells, and that generation of these F1 cytotoxic T lymphocytes (CTL) requires an in vivo priming step in which it is essential that the inoculated parental cells bear the relevant target antigens and possess alloreactivity for F1 host antigens. The requirement for alloreactivity and antigen on the same priming cell population suggests that these potent lytic responses depend on a situation akin to a hapten-carrier effect that bypasses otherwise ineffective helper responses by the host to these unusual antigens. Restimulation of F1 lymphocytes in culture is also necessary, requiring the presence of antigen on irradiated lymphoblast stimulator cells, but alloreactivity to responder cell antigens is not necessary; normal, nonactivated lymph node cells are completely ineffective as stimulators. For effective lysis, the target cells need not possess the potential for alloreactivity to responder F1 CTL. We also demonstrate in a preliminary way additional antigen systems defined by killer populations raised with other F1 antiparental strain combinations.

Polymyositis associated with asymptomatic primary biliary cirrhosis and eosinophilia: An analysis of the mitochondrial antigens by Wsetern blotting.

Polymyositis associated with asymptomatic primary biliary cirrhosis and eosinophilia: An analysis of the mitochondrial antigens by Wsetern blotting.

Primary biliary cirrhosis: Paradigm or paradox for autoimmunity

Primary biliary cirrhosis has been classified as a model autoimmune disease based on striking defects in immune regulation and the presence of autoantibodies to mitochondria. Until recently the significance and definition of mitochondrial autoreactivity was unknown. Since 1987, there has been a vast improvement in the understanding and definition of the biochemical and molecular target autoantigens. The cloning of complementary DNAs for mitochondrial antigens has led to the identification of three enzymes of the 2-oxo-acid dehydrogenase family as the targets of the autoantibodies to mitochondria in patients with primary biliary cirrhosis. The major reactive autoantigen is the E2 subunit of pyruvate dehydrogenase. Immunodominant sites on pyruvate dehydrogenase E2 (autoepitopes) have been mapped and have been shown to be the site of attachment of the functionally important lipoic acid prosthetic group. The autoepitope for the other enzymes probably occupies an equivalent site on the enzyme. The availability and definition of these mitochondrial autoepitopes have allowed specific questions to be addressed relating to the processing and targeting of these autoantigens as well as further studies on mechanisms of immunopathology. Similarly, the availability of well-defined autoantigens could contribute to the development of valid animal models in addition to the already described reproduction of the biliary ductular lesions by transfer of peripheral blood lymphocytes from patients with primary biliary cirrhosis into severe combined immunodeficient mice. Such models will facilitate specific study of the role of major histocompatibility complex expression and the characterization of T-cell reactivity. Thus, primary biliary cirrhosis is a key example of significant progress in autoimmunity being made by use of recombinant DNA technology.