Two types of autoantibody-mediated thrombocytopenia in patients with systemic lupus erythematosus.

OBJECTIVES: To determine whether autoantibodies to two platelet-specific antigens, glycoprotein IIb/IIIa (GPIIb/IIIa) and thrombopoietin receptor (TPOR), contribute to thrombocytopenia in patients with systemic lupus erythematosus (SLE). METHODS: Circulating B cells producing anti-GPIIb/IIIa antibodies and serum anti-TPOR antibodies were measured in 32 SLE patients with thrombocytopenia, 30 SLE patients without thrombocytopenia, 92 patients with idiopathic thrombocytopenia and 60 healthy controls. The megakaryocyte density in bone-marrow smears from all the patients with thrombocytopenia was evaluated. RESULTS: Anti-GPIIb/IIIa and anti-TPOR antibody responses were more frequent in SLE patients with thrombocytopenia than in those without thrombocytopenia (88 vs 17%, P<0.0001; and 22% vs 0%, P=0.01, respectively). The frequencies of these platelet-related antibodies were comparable between SLE patients with thrombocytopenia and patients with idiopathic thrombocytopenia. Twenty-nine (91%) SLE patients with thrombocytopenia had either anti-GPIIb/IIIa or anti-TPOR antibody, and six had both. In SLE patients with thrombocytopenia, the anti-TPOR-positive patients had significantly higher frequencies of megakaryocytic hypoplasia and poorer therapeutic responses to corticosteroids and intravenous immunoglobulin than did the anti-TPOR-negative patients, most of whom had the anti-GPIIb/IIIa antibody alone. CONCLUSIONS: Anti-GPIIb/IIIa and anti-TPOR antibodies are major factors contributing to SLE-associated thrombocytopenia, but the clinical presentations associated with these autoantibodies are different.

Autoantibody to CD40 ligand in systemic lupus erythematosus: association with thrombocytopenia but not thromboembolism.

OBJECTIVES: To examine the prevalence, clinical associations and pathogenic roles of autoantibodies to CD40 ligand (CD40L) in patients with systemic lupus erythematosus (SLE). METHODS: Plasma anti-CD40L antibodies from 125 patients with SLE, 24 with primary antiphospholipid syndrome (APS) and 90 with idiopathic thrombocytopenic purpura (ITP) and from 62 healthy individuals were measured with an enzyme-linked immunosorbent assay (ELISA). HeLa cells transfected with human CD40L cDNA (HeLa/CD40L) were used to confirm the presence of anti-CD40L autoantibodies. The effect of anti-CD40L antibodies on the CD40L-CD40 interaction was evaluated by observing CD40L-induced IkappaB activation in CD40-expressing fibroblasts. RESULTS: Anti-CD40L autoantibody was detected in seven (6%) SLE, three (13%) primary APS and 11 (12%) ITP patients, but in no healthy controls. Antibody binding in an ELISA was competitively inhibited by membrane components of HeLa/CD40L. Anti-CD40L antibody-positive IgG specifically bound the surface of living HeLa/CD40L, as shown by flow cytometry. The frequency of thrombocytopenia was significantly higher in SLE patients with the anti-CD40L antibody than in those without (100 vs 14%; P<0.00001), whereas there was no association between the anti-CD40L antibody and thrombosis. Binding of the anti-CD40L antibodies in patients' plasma to CD40L was competitively inhibited by a series of mouse anti-CD40L monoclonal antibodies. Anti-CD40L antibody-positive IgG failed to inhibit CD40L-induced IkappaB activation. CONCLUSIONS: Anti-CD40L autoantibody is associated with thrombocytopenia but not thromboembolism. Our findings are potentially useful in understanding the complex roles of CD40L in the pathophysiology of thrombosis and haemostasis as well as the thromboembolic complications that occur during treatment with anti-CD40L humanized antibody.

Autoantibodies to the amino-terminal fragment of beta-fodrin expressed in glandular epithelial cells in patients with Sjögren's syndrome.

Sjögrens's syndrome (SS) is an autoimmune disease characterized by destruction of lacrimal and salivary glands, but the mechanisms underlying the disease process are unclear. By immunoscreening a HepG2 cDNA library with serum from an SS patient we isolated a cDNA encoding amino-terminal 616 aa of beta-fodrin, a membrane skeleton protein associated with ion channels and pumps. Serum Ab to the amino-terminal fragment of beta-fodrin was frequently detected in SS patients compared with rheumatic disease patients without SS or healthy controls (70 vs 12 or 4%; p < 0.00001). All the anti-beta-fodrin-positive sera recognized the amino-terminal fragment with no homology to alpha-fodrin. Anti-beta-fodrin Abs in patients' sera as well as mouse polyclonal sera raised against the amino-terminal beta-fodrin fragment did not react with intact beta-fodrin, but recognized the 65-kDa amino-terminal fragment generated through cleavage by caspase-3 or granzyme B. When expression of intact and fragmented beta-fodrin in lacrimal glands was assessed by immunohistochemistry, the antigenic amino-terminal fragment was distributed diffusely in acinar epithelial cell cytoplasm, whereas the carboxyl-terminal fragment and/or intact beta-fodrin were localized in peripheral cytoplasm, especially at the basal membrane, in SS patients. In contrast, intact beta-fodrin was detected primarily at the apical membrane of epithelia, and the amino-terminal fragment was scarcely detected in control patients with chronic graft-vs-host disease. These findings suggest that cleavage and altered distribution of beta-fodrin in glandular epithelial cells may induce impaired secretory function and perpetuate an autoimmune response to beta-fodrin, leading to autoantibody production and glandular destruction in SS.

Autoreactive CD4(+) T-cell clones to beta2-glycoprotein I in patients with antiphospholipid syndrome: preferential recognition of the major phospholipid-binding site.

Autoreactive CD4(+) T cells to beta2-glycoprotein I (beta2GPI) that promote antiphospholipid antibody production were recently identified in patients with antiphospholipid syndrome (APS). To further examine antigen recognition profiles and T-cell helper activity in beta2GPI-reactive T cells, 14 CD4(+) T-cell clones specific to beta2GPI were generated from 3 patients with APS by repeated stimulation of peripheral blood T cells with recombinant beta2GPI. At least 4 distinct T-cell epitopes were identified, but the majority of the beta2GPI-specific T-cell clones responded to a peptide encompassing amino acid residues 276 to 290 of beta2GPI (KVSFFCKNKEKKCSY; single-letter amino acid codes) that contains the major phospholipid-binding site in the context of the DRB4*0103 allele. Ten of 12 beta2GPI-specific T-cell clones were able to stimulate autologous peripheral blood B cells to promote anti-beta2GPI antibody production in the presence of recombinant beta2GPI. T-cell helper activity was exclusively found in T-cell clones capable of producing interleukin 6 (IL-6). In vitro anti-beta2GPI antibody production induced by T-cell clones was inhibited by anti-IL-6 or anti-CD40 ligand monoclonal antibody. In addition, exogenous IL-6 augmented anti-beta2GPI antibody production in cultures of the T-cell clone lacking IL-6 expression. These results indicate that beta2GPI-specific CD4(+) T cells in patients with APS preferentially recognize the antigenic peptide containing the major phospholipid-binding site and have the capacity to stimulate B cells to produce anti-beta2GPI antibodies through IL-6 expression and CD40-CD40 ligand engagement. These findings are potentially useful for clarifying the pathogenesis of APS and for developing therapeutic strategies that suppress pathogenic antiphospholipid antibody production in these patients.

Induction of antigen-specific human CD4(+) T cell anergy by peripheral blood DC2 precursors.

Dendritic cells (DC) are antigen (Ag)-presenting cells that are essential for initiation of T cell-dependent immunity, and distinct DC subsets are known to direct different classes of immune responses. DC2 precursors (pDC2) or plasmacytoid DC were recently identified as a Th2-skewing and IFN-alpha-producing human DC subset. Here, we demonstrate that pDC2 enriched from human peripheral blood have a capacity to induce an anergic state in human Ag-specific CD4(+) T cell lines. Tetanus toxoid-specific T cell lines incubated with tetanus toxoid-pulsed autologous pDC2 failed to proliferate in secondary cultures with optimal Ag stimulation. T cell anergy induction required TCR engagement with Ag/MHC complex presented on pDC2. T cells rendered anergic lost IL-2 production but produced IFN-gamma and IL-10 upon stimulation. The pDC2-induced unresponsiveness was completely or partially reversible when a high concentration of exogenous IL-2 was added in the secondary cultures. Autoreactive CD4(+) T cell clones specific for topoisomerase I derived from a patient with scleroderma were also rendered anergic after co-culture with topoisomerase I-pulsed autologous pDC2,resulting in failure to proliferate or provide help to B cells. These results suggest that pDC2 are involved in maintenance of peripheral T cell tolerance and have potential for use in the suppression of pathogenic T cell responses in autoimmune diseases and organ transplantation.

An immunodominant epitope on DNA topoisomerase I is conformational in nature: heterogeneity in its recognition by systemic sclerosis sera.

OBJECTIVE: To characterize an immunodominant epitope recognized by anti-DNA topoisomerase I (topo I) antibody, a major autoantibody in sera of patients with systemic sclerosis (SSc). METHODS: Topo I fragments were generated as fusion proteins using a bacterial expression system as well as polypeptides translated in vitro using a eukaryotic expression system. Reactivities to the 2 preparations of recombinant topo I polypeptides in anti-topo I-positive sera from SSc patients of varied ethnic backgrounds were examined by immunoblotting, immunoprecipitation, and/or enzyme-linked immunosorbent assay. RESULTS: The fragment encoding amino acids 489-573 of topo I was recognized by 98 of 100 anti-topo I-positive SSc sera. Both carboxyl- and amino-terminal deletion studies as well as competitive inhibition assays using topo I synthetic peptides showed that a region of > or =52 amino acids (512-563) was necessary for recognition by anti-topo I antibodies. The minimum epitope region and conformation required for this reactivity were variable among sera from Caucasian, African American, Japanese, and Choctaw SSc patients. CONCLUSION: An immunodominant epitope recognized by anti-topo I autoantibody is located in the region of amino acids 489-573 of the topo I protein and is largely conformational in nature. The recognition pattern of this region by anti-topo I-positive sera is heterogeneous and is influenced by ethnic background.

Coexistence of serum anti-DNA topoisomerase I and anti-Sm antibodies: report of 3 cases.

We describe 3 Japanese patients having both serum anti-DNA topoisomerase I and anti-Sm antibodies. All 3 patients had typical features of systemic lupus erythematosus, such as glomerulonephritis, in addition to skin thickening and systemic sclerosis related organ involvement, including pulmonary interstitial fibrosis and renal crisis. This is the first report of the coexistence of these 2 disease specific autoantibodies.

Nodular regenerative hyperplasia of the liver in a patient with systemic sclerosis.

We report on a 33-year-old female patient with systemic sclerosis and nodular regenerative hyperplasia of the liver (NRHL). A needle biopsy of the patient's liver did not reveal the histology of NRHL or liver cirrhosis at her first visit to our hospital, when portal hypertension was demonstrated by percutaneous transhepatic portography. After 11 years, the patient died of hepatic and renal failure. At the time of autopsy, multiple nodules were found in the liver, and a microscopic examination showed a histology compatible with NRHL. It is suggested that the immunological disturbance was related to the patient's portal hypertension and NRHL.

Enhancement of anti-DNA topoisomerase I autoantibody response after lung cancer in patients with systemic sclerosis. A report of two cases.

Anti-DNA topoisomerase I (anti-topo I) antibody profiles were compared before and after lung cancer in 2 patients with systemic sclerosis (SSc). Both patients developed adenocarcinoma of the lung late in the course of SSc and died of the cancer. Anti-topo I antibody levels, determined by double immunodiffusion and enzyme-linked immunosorbent assay, increased markedly at the time of diagnosis of lung cancer. Furthermore, patients' sera obtained after lung cancer reacted with multiple epitopes on the entire topo I molecule, some of which had not previously been recognized. These results further support the concept that anti-topo I antibody production in SSc patients is due to an antigen-driven process.

The presence of an antigen reactive with a human autoantibody in Trichosia pubescens (Diptera: Sciaridae) and its association with certain transcriptionally active regions of the genome.

The antigens in HeLa and Trichosia pubescens cells, recognized by sera from patients with rheumatic diseases containing anti-Ku antibodies, were compared by means of immunoprecipitation of labeled cell extracts. The autoantibodies present in the tested sera precipitate at least two polypeptides of approximately Mr = 70,000 and Mr = 80,000 in HeLa cell extracts and a polypeptide of approximately Mr = 72,000 in Trichosia salivary gland cell extracts. The distribution of the insect antigen in chromatin was studied in salivary gland polytene chromosomes by indirect immunofluorescent staining with sera from two different patients. Both sera react with certain transcriptionally active chromosomal sites. The presence of the antigen in polytene chromosomes is strictly dependent on transcription, as no reaction is observed in the same sites before or after gene activation. Other sites, such as the nucleolar organizing region, are very active in transcription but never reacted with the anti-Ku positive sera. These results show that the insect antigen is associated with transcription-related processes of a subset of the chromosomal loci of T. pubescens. The anti-Ku positive sera react with a highly conserved antigen, which may serve a very important and similar role in the cellular metabolism of both insect and mammalian cells.

Identification of human anti-DNA, anti-RNP, anti-SM, and anti-SS-A serum antibodies bearing the cross-reactive 16/6 idiotype.

The human idiotype 16/6, characteristic of lupus-derived anti-DNA autoantibodies, was detected in sera of Japanese patients with autoimmune diseases. The 16/6-positive sera were from patients with active SLE and, in one case, a scleroderma-polymyositis overlap syndrome; all contained anti-ssDNA antibody. Affinity chromatography was used to isolate 16/6-bearing Ig from sera of seven patients having anti-DNA and other autoantibodies. In a preliminary step, rheumatoid factors and nonspecific binding were removed by four passes of the serum through a column of immobilized normal rabbit gamma-globulin. The absorbed sera were then applied to a column of immobilized specific rabbit anti-16/6 globulin. This combined procedure depleted anti-ssDNA activity selectively. From 44 to 60% of total Ig but only 6 to 25% of anti-ssDNA activity was recovered in the material that passed through both columns. Approximately 40% of the anti-RNP/Sm and anti-SS-A/SS-B activities were recovered, reflecting a lesser degree of selective depletion of these antibodies. The specific eluate from the anti-idiotype column contained more IgG than IgM; anti-DNA activity; anti-RNP, anti-Sm, or anti-SS-A activity. Soluble DNA inhibited the binding of one eluate to RNP/Sm but not of another eluate to SS-A/SS-B. The results confirm that the genes encoding the 16/6 idiotype are widely represented in ethnically different human populations, and indicate that 16/6-bearing Ig may bind to other autoantigens as well as to DNA.

Parallel sets of autoantibodies in MRL-lpr/lpr mice. An anti-DNA, anti-SmRNP, anti-gp70 network.

The public idiotype Id-H130 occurs in MRL-lpr/lpr serum both on a high proportion of anti-DNA autoantibodies as well as on antibodies that do not bind to DNA. To define members of the latter population, we prepared hybridomas and selected Id-H130+ mAbs that did not bind to DNA. One such antibody, mAb 28/12, was found to be an anti-SmRNP antibody. To determine whether mAb 28/12 had rheumatoid factor activity, we tested its ability to bind, in a solid-phase assay, to 16 mouse IgM mAbs. mAb 28/12 bound to only four of the panel, two anti-DNA antibodies (mAbs 512 and 319) and two anti-gp70 antibodies (mAbs 514 and 1417). In a liquid-phase competition assay with a panel of 32 monoclonal IgM and IgG antibodies, including allotype-matched Igs, mAb 28/12 reacted only with mAbs 512, 319, 514, and 1417. The binding of mAb 28/12 to mAbs 512 and 319 was displaced by DNA, but not by RNA, indicating that the idiotype it defines (Id-28/12) is in the antigen-binding region of the two anti-DNA antibodies. In the two anti-gp70 antibodies (mAbs 514 and 1417), Id-28/12 seems to occur in the framework region. To determine if all four Id-28/12+ antibodies shared a common antigen-binding property, they were tested for their ability to react with DNA and gp70. The two anti-gp70 antibodies did not bind to DNA. However, the two anti-DNA antibodies were found to immunoprecipitate viral proteins from retrovirus-infected cells. mAb 512 reacted with gp70, both in cell membrane lysates and in purified form; mAb 319 reacted with gp85, which contains both gp70 and the retroviral protein p15. Antibodies with properties similar to those of mAb 28/12 were found in MRL-lpr/lpr serum. It was possible, by affinity chromatography on an anti-gp70 antibody column, to isolate from serum those anti-(anti-gp70) antibodies with anti-SmRNP activity. These results show that parallel sets of autoantibodies, which share a common idiotype, but which bind to different autoantigens, occur in MRL-lpr/lpr mice. Some populations of anti-DNA, anti-SmRNP, and anti-gp70 antibodies appear to constitute a network of autoantibodies in that strain. We speculate that part of the anti-SmRNP population of autoantibodies can arise by mutation of germline-encoded anti-DNA antibodies.