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1.
Ann N Y Acad Sci ; 998: 237-56, 2003 Sep.
Article in English | MEDLINE | ID: mdl-14592881

ABSTRACT

We have studied responses in thymoma patients to interferon-alpha and to the acetylcholine receptor (AChR) in early-onset myasthenia gravis (EOMG), seeking clues to autoimmunizing mechanisms. Our new evidence implicates a two-step process: (step 1) professional antigen-presenting cells and thymic epithelial cells prime AChR-specific T cells; then (step 2) thymic myoid cells subsequently provoke germinal center formation in EOMG. Our unifying hypothesis proposes that AChR epitopes expressed by neoplastic or hyperplastic thymic epithelial cells aberrantly prime helper T cells, whether generated locally or infiltrating from the circulation. These helper T cells then induce antibody responses against linear epitopes that cross-react with whole AChR and attack myoid cells in the EOMG thymus. The resulting antigen-antibody complexes and the recruitment of professional antigen-presenting cells increase the exposure of thymic cells to the infiltrates and provoke local germinal center formation and determinant spreading. Both these and the consequently enhanced heterogeneity and pathogenicity of the autoantibodies should be minimized by early thymectomy.


Subject(s)
Autoimmunity , B-Lymphocytes/immunology , Myasthenia Gravis/immunology , T-Lymphocytes/immunology , Age of Onset , Animals , Autoantibodies , Bungarotoxins/metabolism , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/physiology , Epitopes/immunology , Fluorescent Antibody Technique , Germinal Center , Histocompatibility Antigens Class II/metabolism , Humans , Insulin/metabolism , Interferon-alpha/immunology , Interleukin-2/immunology , Keratins/metabolism , Models, Immunological , Mutation , Myasthenia Gravis/metabolism , Receptors, Cholinergic/immunology , Stromal Cells , T-Lymphocytes/classification , Thymoma/immunology , Thymus Gland/cytology , Thymus Gland/physiology , Thymus Neoplasms , Troponin I/metabolism
2.
J Immunol ; 167(4): 1935-44, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11489973

ABSTRACT

The muscle weakness in myasthenia gravis (MG) is mediated by autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Production of these pathogenic autoantibodies is believed to be associated with germinal centers (GC) and anti-AChR-secreting plasma cells in the hyperplastic thymus of patients with early onset MG (EOMG). Here, we describe the repertoire of rearranged heavy chain V genes and their clonal origins in GC from a typical EOMG patient. Three hundred fifteen rearranged Ig V(H) genes were amplified, cloned, and sequenced from sections of four thymic GC containing AChR-specific B cells. We found that thymic GC contain a remarkably heterogeneous population of B cells. Both naive and circulating memory B cells undergo Ag-driven clonal proliferation, somatic hypermutation, and selection. Numerous B cell clones were present, with no individual clone dominating the response. Comparisons of B cell clonal sequences from different GC and known anti-AChR Abs from other patients showed convergent mutations in the complementarity determining regions. These results are consistent with AChR driving an ongoing GC response in the thymus of EOMG patients. This is the first detailed analysis of B cell clones in human GC responding to a defined protein Ag, and the response we observed may reflect the effects of chronic stimulation by autoantigen.


Subject(s)
B-Lymphocyte Subsets/metabolism , Gene Rearrangement, B-Lymphocyte, Heavy Chain/genetics , Germinal Center/metabolism , Mutation , Myasthenia Gravis/genetics , Myasthenia Gravis/immunology , Receptors, Cholinergic/immunology , Thymus Gland/metabolism , Adult , Amino Acid Sequence , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/pathology , Bungarotoxins/metabolism , Clone Cells , Complementarity Determining Regions/biosynthesis , Complementarity Determining Regions/genetics , Female , Gene Amplification , Germinal Center/cytology , Germinal Center/immunology , Germinal Center/pathology , Humans , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Immunologic Memory/genetics , Interphase/genetics , Interphase/immunology , Iodine Radioisotopes/metabolism , Lymphocyte Activation/genetics , Lymphoid Tissue/cytology , Molecular Sequence Data , Multigene Family/immunology , Receptors, Cholinergic/metabolism , Thymus Gland/cytology , Thymus Gland/immunology , Thymus Gland/pathology , Tumor Cells, Cultured
5.
Lupus ; 8(1): 29-38, 1999.
Article in English | MEDLINE | ID: mdl-10025597

ABSTRACT

We have investigated the effects of interleukin-2 (IL-2) and transforming growth factor-beta (TGF-beta) gene therapy on the progress of autoimmune disease in MRL-lpr/lpr mice, a murine model of systemic lupus erythematosus (SLE). These mice have uncontrolled proliferation of T cells, an impaired response to T cell mitogen and produce autoantibodies against nuclear antigens, including DNA. Immune complexes formed by these autoantibodies are believed to cause glomerulonephritis and vasculitis in lupus mice and human SLE. Since there is an imbalance of cytokine production in both SLE patients and lupus mice, we examined the effects of cytokine gene therapy on the progression of autoimmune disease in MRL-lpr/lpr mice. The mice were treated orally with a non-pathogenic strain of Salmonella typhimurium bearing the aroA-aroD- mutations and carrying the murine genes encoding IL-2 and TGF-beta. The bacteria synthesise and slowly release the cytokines in vivo. Our results show that, contrary to expectation, TGF-beta gene therapy produced no improvement in pathology and generally had opposite effects to those of IL-2. IL-2 gene therapy restored the defective T cell proliferative response to mitogen and suppressed the autoantibody response, glomerulonephritis and growth of lymphoid tumours.


Subject(s)
Genetic Therapy , Interleukin-2/genetics , Lupus Erythematosus, Systemic/therapy , Salmonella typhimurium/genetics , Transforming Growth Factor beta/genetics , Animals , Antibodies, Antinuclear/biosynthesis , Female , Kidney/pathology , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/pathology , Lymphocyte Activation , Mice , Mice, Inbred MRL lpr , T-Lymphocytes/immunology
6.
J Clin Invest ; 102(5): 938-46, 1998 Sep 01.
Article in English | MEDLINE | ID: mdl-9727062

ABSTRACT

Structures resembling germinal centers are seen in the salivary glands of patients with Sjögren's syndrome, but it is not known whether the microenvironment of these cell clusters is sufficient for the induction of a germinal center response. Therefore, we cloned and sequenced rearranged Ig V genes expressed by B cells isolated from sections of labial salivary gland biopsies from two Sjögren's syndrome patients. Rearranged V genes from B cells within one cell cluster were polyclonal and most had few somatic mutations. Two adjacent clusters from another patient each contained one dominant B cell clone expressing hypermutated V genes. None of the rearranged V genes was found in both clusters, suggesting that cells are unable to migrate out into the surrounding tissue and seed new clusters. The ratios of replacement to silent mutations in the framework and complementarity determining regions suggest antigen selection of high-affinity mutants. These results show that an antigen-driven, germinal center-type B cell response is taking place within the salivary glands of Sjögren's syndrome patients. In view of the recent demonstration of a germinal center response within the rheumatoid synovial membrane and the existence of similar structures in the target tissues of other autoimmune diseases, we propose that germinal center- type responses can be induced in the nonlymphoid target tissues of a variety of autoimmune diseases.


Subject(s)
Antigens/immunology , Autoimmune Diseases/immunology , B-Lymphocytes/metabolism , Salivary Glands/immunology , Sjogren's Syndrome/immunology , Cell Division/immunology , Clone Cells/immunology , Cloning, Molecular , Germinal Center/immunology , Humans , Immunoglobulin Variable Region/genetics , Immunohistochemistry , Salivary Glands/cytology , Sequence Alignment , Sequence Analysis, DNA
7.
Hybridoma ; 17(3): 299-304, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9708833

ABSTRACT

Human hybridomas that secrete monoclonal antibodies (MAbs) in a stable manner are technically difficult to obtain. The problems limiting their production are the low numbers of sensitized B cells in peripheral blood, the limited choice of techniques for B cell immortalization, the limited number of suitable human myeloma or lymphoblastoid fusion partners, and the inability to immunize humans with most antigens. In order to circumvent these problems, we have compared the efficiency of different methods for production of B cell lines secreting human MAbs against the nuclear antigens dsDNA, ssDNA, and Sm/RNP from patients with the autoimmune disease systemic lupus erythematosus (SLE). We have tested various combinations of the following procedures: (1) EBV infection for immortalization of activated B lymphocytes, (2) activation of human resting B lymphocytes by anti-CD40, and (3) direct fusion of lymphocytes with a myeloma cell line using PBL or splenocytes from SLE patients. The methodological aspects of this investigation include optimization of the CD40 system and the generation of human hybridomas specific for nuclear antigens by fusion between sensitized lymphocytes and the human/mouse heteromyeloma cell line CBF7. The most efficient method for producing stable, IgG autoantibody-secreting human hybridomas was fusion of lymphocytes with cell line CBF7; human spleen was the best source of lymphocytes.


Subject(s)
Antibodies, Antinuclear/immunology , Autoantibodies/immunology , Hybridomas/immunology , Antibodies, Antinuclear/biosynthesis , Antibodies, Antinuclear/isolation & purification , Autoantibodies/biosynthesis , Autoantibodies/isolation & purification , B-Lymphocytes/immunology , B-Lymphocytes/pathology , Cell Line/immunology , Humans , Lupus Erythematosus, Systemic/immunology , Lymphocyte Activation
9.
J Exp Med ; 183(4): 1447-59, 1996 Apr 01.
Article in English | MEDLINE | ID: mdl-8666903

ABSTRACT

MRL/MP-lpr/lpr (MRL/lpr) mice develop a spontaneous autoimmune disease. Serum from these mice contained significantly higher concentrations of nitrite/nitrate than serum from age-matched control MRL/MP-+/+ (MRL/+), BALB/c or CBA/6J mice. Spleen and peritoneal cells from MRL/lpr mice also produced significantly more nitric oxide (NO) than those from the control mice when cultured with interferon (IFN) gamma and lipopolysaccharide (LPS) in vitro. It is interesting to note that peritoneal cells from MRL/lpr mice also produced markedly higher concentrations of interleukin (IL) 12 than those from MRL/+ or BALB/c mice when cultured with same stimuli. It is striking that cells from MRL/lpr mice produced high concentrations of NO when cultured cells from MRL/+ or BALB/c mice. The enhanced NO synthesis induced by IFN-gamma/LPS was substantially inhibited by anti-IL-12 antibody. In addition, IL-12-induced NO production can also be markedly inhibited by anti-IFN-gamma antibody, but only weakly inhibited by anti-tumor necrosis factor alpha antibody. The effect of IL-12 on NO production was dependent on the presence of natural killer and possibly T cells. Serum from MRL/lpr mice contained significantly higher concentrations of IL-12 compared with those of MRL/+ or BALB/c control mice. Daily injection of recombinant IL-12 led to increased serum levels of IFN-gamma and NO metabolites, and accelerated glomerulonephritis in the young MRL/lpr mice (but not in the MRL/+ mice) compared with controls injected with phosphate-buffered saline alone. These data, together with previous finding that NO synthase inhibitors can ameliorate autoimmune disease in MRL/lpr mice, suggest that high capacity of such mice to produce IL-12 and their greater responsiveness to IL-12, leading to the production of high concentrations of NO, are important factors in this spontaneous model of autoimmune disease.


Subject(s)
Glomerulonephritis/etiology , Interleukin-12/metabolism , Lupus Erythematosus, Systemic/etiology , Nitric Oxide/metabolism , Animals , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Interferon-gamma/metabolism , Interleukin-12/genetics , Interleukin-12/pharmacology , Kidney/pathology , Killer Cells, Natural/metabolism , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Mice, Mutant Strains , Models, Biological , Peritoneum/cytology , Peritoneum/metabolism , Recombinant Proteins/pharmacology , Spleen/cytology , Spleen/metabolism , T-Lymphocytes/metabolism
10.
Lupus ; 4(4): 297-303, 1995 Aug.
Article in English | MEDLINE | ID: mdl-8528227

ABSTRACT

In serum and plasma from SLE patients, we have detected elevated levels of factors which regulate proliferative responses of CTLL cells to IL-2. Serum samples containing these factors have dose-dependent dual inhibitory and stimulatory activities on the proliferation of this IL-2-dependent T lymphocyte cell line. At high concentrations, the serum factors inhibit the proliferative responses of CTLL cells to IL-2. At low concentrations, they synergise with IL-2 stimulating the growth of cells. Similar inhibitory activity, but with lower titre, was also found to be elevated in sera of some MRL/lpr mice, an animal model of SLE. Functional characterisation of the serum factors shows that: (1) the inhibitory activity cannot be neutralised by exogenous IL-2; (2) the stimulatory activity is not due to the presence of serum IL-2 but synergy of the factor with IL-2; (3) the factors bind directly to CTLL cells but they do not bind to protein A; and (4) the serum factors are not dialysable but heat labile. The possible pathological implications of the serum factors, particularly for the defective T cell functions in lupus disease, are discussed.


Subject(s)
Biological Factors/blood , Interleukin-2/blood , Lupus Erythematosus, Systemic/blood , T-Lymphocytes/physiology , Animals , Cell Division , Cell Line , Disease Models, Animal , Humans , Lupus Erythematosus, Systemic/immunology , Mice , Mice, Inbred BALB C , Staphylococcal Protein A/blood , T-Lymphocytes/cytology
11.
Autoimmunity ; 15(1): 19-29, 1993.
Article in English | MEDLINE | ID: mdl-8218827

ABSTRACT

Splenic T-cells from lupus strain (NZB/W F1, Mrl/lpr) mice lack the ability to respond to concanavalin A (Con A) by secretion of IL-2 and hence expression of IL-2 receptor and proliferation. These defects were found not only in an aged group (> 5 months) of mice in which obvious clinical 'SLE like' symptoms and elevated levels of serum autoantibodies were observed, but also in mice as young as 4-wk. We demonstrate here that the defective mitogenic activation of T-cells from lupus mice is due to the inability of T-helper cells to produce IL-2 and this defect can be restored by exogenous IL-2 in vitro. Con A-induced cell proliferation and IL-2 receptor expression on CD3+ cells from lupus mice occur only in the presence of exogenous IL-2, whereas normal T-cells from BALB/c and CBA control mice are activated by the mitogen and undergo complete cell cycling in the absence of exogenous IL-2, as they are able to secrete sufficient endogenous IL-2. The detection of impaired T-helper function in young lupus mice, before development of overt disease, and the reversible nature of the defect indicate that defective IL-2 activity may be fundamental to the mechanism of development of pathology in SLE.


Subject(s)
Autoimmune Diseases/immunology , Interleukin-2/pharmacology , Lupus Erythematosus, Systemic/immunology , Lymphocyte Activation/drug effects , Mice, Inbred NZB/immunology , T-Lymphocyte Subsets/drug effects , T-Lymphocytes, Helper-Inducer/immunology , Age Factors , Animals , Autoimmune Diseases/genetics , Cells, Cultured , Concanavalin A/pharmacology , Female , Interleukin-2/deficiency , Interleukin-2/metabolism , Lupus Erythematosus, Systemic/genetics , Mice , Mice, Inbred BALB C/immunology , Mice, Inbred CBA/immunology , Receptors, Interleukin-2/analysis , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Helper-Inducer/drug effects , T-Lymphocytes, Helper-Inducer/metabolism
12.
Autoimmunity ; 12(4): 249-58, 1992.
Article in English | MEDLINE | ID: mdl-1391594

ABSTRACT

We have investigated the number of B-lymphocyte clones secreting anti-ssDNA antibodies in SLE patients and a chronic active hepatitis patient by isoelectric focusing and reverse immunoblotting of serum antibodies. Individual clones can be identified by the unique pattern of bands produced by their antibodies (the clonotype). Using this technique, we have shown that the anti-DNA response of the majority of SLE patients is clonally restricted, in many cases only a single B-cell clone responding. We have also measured qualitative and quantitative changes in expression of B-cell clones and shown that these clones are remarkably stable with lifespans of up to six years or more. These results are in agreement with previous observations of clonal restriction of the anti-DNA response in three mouse models of SLE and in addition show that, unlike the mouse models, human anti-DNA-secreting B-cell clones are extremely stable and long-lived. The implications of these results for models of initiation and regulation of the autoimmune response are discussed.


Subject(s)
Antibodies, Antinuclear/analysis , B-Lymphocytes/immunology , Lupus Erythematosus, Systemic/immunology , Cell Survival , Clone Cells , DNA, Single-Stranded/immunology , Humans
14.
Autoimmunity ; 8(2): 159-68, 1990.
Article in English | MEDLINE | ID: mdl-2104188

ABSTRACT

We present the results of a study of the physical, haematological and serological features of the progress of the SLE-like syndrome in MRL/Mp-lpr/lpr and (NZB x NZW)Fl mice. As part of this study, we have analysed the IEF spectrotypes of anti-ssDNA antibodies in the sera of these mice and shown that the anti-ssDNA response is clonally restricted, as we have previously shown in a mouse chimaera model and in human SLE. Sequential qualitative and quantitative analysis of anti-ssDNA clonotypes has revealed that the lupus mouse anti-ssDNA clones are relatively short lived, having a lifespan of only 6 to 8 weeks, contrasting sharply with the much longer lifespan previously reported for a mouse anti-DNP-secreting clone and the exceptionally long lifespan of most anti-ssDNA-secreting clones of SLE patients. The implications of these observations for our understanding of the regulation of the autoimmune response are discussed.


Subject(s)
Antibodies, Antinuclear/analysis , B-Lymphocytes/immunology , DNA, Single-Stranded/immunology , Lupus Erythematosus, Systemic/immunology , Age Factors , Animals , Clone Cells/immunology , Disease Models, Animal , Female , Immunoglobulin G/analysis , Immunoglobulin M/analysis , Isoelectric Focusing , Male , Mice , Mice, Inbred Strains
16.
Br J Ophthalmol ; 73(2): 155-9, 1989 Feb.
Article in English | MEDLINE | ID: mdl-2930762

ABSTRACT

The antibody responses to human and bovine retinal S antigen in the sera of patients with uveitis from various causes were compared with those of a group of healthy volunteers who were fully screened for signs of eye disease. Antiretinal antibodies were found with equal frequency and through the same range of titres in patients and controls, irrespective of the nature or activity of the uveitis. These findings were confirmed by spectrotypic analysis of sera from patients and controls where the predominant serum antibody response was polyclonal. In a small group of patients with retinal vasculitis there was an additional monoclonal response, indicating clonal expansion of a single lymphocyte subset. The prevalence of serum antibodies to retinal antigens in the normal population may indicate a protective role for 'natural' autoantibodies, as has recently been suggested for autoimmune diseases generally.


Subject(s)
Antigens, Protozoan/immunology , Autoantibodies/biosynthesis , Autoantigens/immunology , Uveitis/immunology , Animals , Cattle , Humans , Isoelectric Focusing , Retinal Diseases/immunology , Retinal Vessels , Uveitis, Anterior/immunology
17.
Clin Exp Immunol ; 73(2): 269-75, 1988 Aug.
Article in English | MEDLINE | ID: mdl-3052944

ABSTRACT

We have studied the isoelectric focusing (IEF) spectrotypes of autoantibodies against thyroglobulin in two rat models of Hashimoto's thyroiditis: (1) AUG strain rats immunized with autologous thyroglobulin in Freund's complete adjuvant; (2) PVG/c strain rats which have been thymectomized and sublethally irradiated. The IEF spectrotypes revealed differences between the two models. The anti-thyroglobulin response in immunized AUG rats is mainly oligoclonal or polyclonal, often with dominant clones in the spectrotype, similar to about 90% of Hashimoto's patients, whereas the response in the PVG/c rat is highly restricted. There were pronounced changes in spectrotype with time in the PVG/c, but not AUG, rats with considerable variation in the lifespan of individual clones. The maximum lifespan of an anti-self secreting B-cell clone in the PVG/c rat, determined by persistence of its clonotype, was at least 16 weeks.


Subject(s)
Autoantibodies/classification , Disease Models, Animal , Thyroglobulin/immunology , Thyroiditis, Autoimmune/immunology , Animals , Autoantibodies/biosynthesis , Clone Cells/immunology , Female , Isoelectric Focusing , Rats , Rats, Inbred Strains , Thymectomy , Thyroiditis, Autoimmune/etiology , Time Factors , Whole-Body Irradiation
18.
J Immunol Methods ; 107(1): 153, 1988 Feb 24.
Article in English | MEDLINE | ID: mdl-3343517
19.
Autoimmunity ; 1(4): 253-66, 1988.
Article in English | MEDLINE | ID: mdl-2980966

ABSTRACT

BALB/c mice neonatally injected with semiallogeneic (C57BL/6 x BALB/c) F1 spleen cells become tolerant to C57BL/6 alloantigens and exhibit chimaerism due to persistence of F1 lymphocytes. Such mouse chimaeras develop an autoimmune (lupus-like) disease characterised by hypergammaglobulinaemia with production of autoantibodies against DNA, Sm antigen and other self-antigens characteristic of SLE in addition to circulating immune complexes and glomerular deposition of immunoglobulins. We have studied the autoimmune response by analysing the isoelectric focusing (IEF)+ patterns (spectrotypes) of anti-ss and anti-dsDNA antibodies produced by these animals. The results show that the anti-DNA response is remarkably restricted, only a very small number of lymphoid cell clones responding in the majority of animals. The behaviour of these clones has been followed during the development of the autoimmune response by analysis of their individual IEF patterns (clonotypes). The first appearance of clones secreting anti-DNA autoantibodies was observed in 3-4 week old mice. Changes in spectrotype occurred during the course of the response but they remained restricted to a very small number of clones in almost all the animals studied. Changes in clonotype consistent with somatic mutation in committed, anti-DNA-secreting clones were also observed. Helper T-lymphocytes of host origin are shown to be required for the development of an autoimmune response.


Subject(s)
Antibodies, Antinuclear/biosynthesis , Autoimmunity/physiology , DNA/immunology , Immune Tolerance/immunology , Isoantigens/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Chimera/immunology , Disease Models, Animal , Lupus Erythematosus, Systemic/immunology , Mice , Mice, Inbred BALB C
20.
Br J Haematol ; 64(4): 745-50, 1986 Dec.
Article in English | MEDLINE | ID: mdl-3801321

ABSTRACT

Immuno-isoelectric focusing (IIEF), a technique previously shown to be sensitive for the detection of paraproteinaemia, was used to test 200 individuals over the age of 45, without history of B cell neoplasm, for the presence of serum paraproteinaemia. 11% of these individuals had evidence of paraproteinaemia detectable by IIEF compared with only 2% by zonal and immunoelectrophoresis. A further 12% had oligoclonal immunoglobulins and the remainder had no qualitative abnormality of the immunoglobulin profile. These results are discussed with particular reference to the aetiology, diagnosis and monitoring of potential B cell neoplasm in high risk individuals or groups.


Subject(s)
Paraproteinemias/epidemiology , Aged , Aged, 80 and over , Humans , Isoelectric Focusing , Middle Aged , Paraproteinemias/diagnosis , Scotland
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