The role of interleukin 12 and nitric oxide in the development of spontaneous autoimmune disease in MRL/MP-lpr/lpr mice.

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.

Antigen-driven clonal proliferation of B cells within the target tissue of an autoimmune disease. The salivary glands of patients with Sjögren's syndrome.

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.

Nonhistone chromatin proteins of B-lymphocytes stimulated by lipopolysaccharide. Two-dimensional gel electrophoresis analysis of proteins synthesized and phosphorylated during the initiation of lymphocyte differentiation.

Synthesis and phosphorylation of nonhistone chromatin and nucleoplasmic proteins during the first 24 h of activation of mouse B-lymphocytes by the B-cell mitogen lipopolysaccharide have been studied by two-dimensional gel electrophoretic analysis. Although little change occurs in the nucleoplasmic proteins, it has been shown that the incorporation of [35S]methionine into nonhistone chromatin proteins is selectively stimulated. The degree of stimulation and the kinetics of synthesis are characteristic for each individual protein; some proteins exhibit increased incorporation only 4 h after addition of mitogen, while others are synthesized de novo between 8 and 24 h. After 72 h stimulation, the majority of nonhistone chromatin protein synthesis occurs in the highly differentiated lymphoblasts and plasma cells actively secreting IgM, very little synthesis taking place in the small lymphocytes. Analysis of nuclear proteins from lymphocytes stimulated for 2 h showed no selective stimulation of phosphorylation. These observations suggest that nonhistone chromatin proteins play an important role in the regulation of gene expression in B-lymphocytes.

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide. III. De novo synthesis.

Nuclear and cytoplasmic proteins synthesised by mouse lymphocytes stimulated in vitro by the B lymphocyte mitogen, lipopolysaccharide, have been analysed by one- and two-dimensional polyacrylamide gel electrophoresis at early and later times after the onset of stimulation. During the first 4 h no change was observed in the electrophoretic profiles but differences in turnover of various nuclear proteins within the same sample were noted. This is contrasted with the previous observations that phosphorylation of nuclear proteins is stimulated within 2 h. (Stott, D.I. and Williamson, A.R. (1978) Biochim. Biophys. Acta 521, 739-752). During prolonged culture, synthesis of nucleoplasmic proteins declined between day 2 and day 3, being reduced to undetectable levels at the end of the 3-day culture period. In contrast, synthesis of many non-histone chromatin proteins was stimulated between 24 and 48 h, the time course and degree of stimulation varying between different proteins. Certain proteins appeared to be synthesized de novo. These events occur at a time of rapidly increasing IgM synthesis and cell differentiation. It is suggested that an initial step in B lymphocyte triggering may involve phosphorylation of preexisting nuclear proteins leading to gene activation followed by synthesis and phosphorylation of new gene regulatory molecules.

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide.

The synthesis of non-histone chromatin proteins and nucleoplasmic proteins has been followed during lipopolysaccharide-induced division and differentiation of murine B lymphocytes. Synthesis was measured by pulse labelling with [3H]leucine, extraction of proteins was under conditions designed to prevent proteolysis and analysis of labelled proteins was by polyacrylamide gel electrophoresis. The average specific activity of non-histone chromatin proteins increased 3-fold, to a maximum, after culture for 24 h with lipopolysaccharide. Comparison of the relative synthesis of individual proteins (stimulation index) reveals three distinct responses: (1) those in the largest group show low stimulation indices, generally less than two; (2) a group of four proteins have indices between 4 and 5; (3) two proteins (molecular weights 21 000 and 22 000) both show an index of 5 at 24 h rising to between 7 and 8 by 48 h when the average specific activity is falling, coinciding with the period of rapid differentiation to high rate IgM secretion. Additionaly at this time, a newly labelled protein (Mr = 36 500) appears in the nucleoplasm followed by a second protein (Mr = 63 000) appearing between 48 and 72 h. The patterns of change are consistent with an overall increase in non-histone chromatin proteins synthesis, necessary for cell division, with superimposed specific changes in synthesis of non-histone chromatin proteins which could be related to regulation of cell differentiation.

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide. II. Phosphorylation.

Stimulation of mouse lymphocytes with the B lymphocyte specific mitogen lipopolysaccharide results in an increased rate of phosphorylation of non-histone chromatin proteins. An initial small increase in phosphorylation occurs during the first 2 h and a much larger increase after 24 h of culture with mitogen. The phosphorylated nuclear and cytoplasmic proteins were analysed by polyacrylamide gel electrophoresis and the stimulation index of each prominent peak measured. It was inferred that selective stimulation of the phosphorylation of individual proteins had occurred from: (1) the range of stimulation indices for different proteins, and (2) the appearance, after 8 h stimulation of an apparently newly phosphorylated non-histone chromatin protein of molecular weight 115 000. The pool size of ATP was monitored and showed only small changes during the first 24 h of exposure to lipopolysaccharide. Phosphatase activity was found to be associated with lymphocyte chromatin and nucleoplasm and may help to regulate the level of phosphorylation of non-histone chromatin proteins in vivo. To preserve phosphorylated proteins during their isolation phosphatase activity was inhibited by Na2MoO4. The selective changes in phosphorylation of nuclear proteins precede, and continue during, the stimulation of immunoglobulin and DNA synthesis. Our results are thus consistent with the hypothesis that phosphorylation of non-histone chromatin proteins plays a role in the regulation of gene expression in B lymphocytes.

Nuclear proteins associated with gene expression in mouse plasmacytoma cell lines.

Nucleoplasmic and non-histone chromatin proteins from two unrelated and four related mouse plasmacytoma cell lines have been analysed by biosynthetic labelling with [35S]-methionine followed by one- and two-dimensional polyacrylamide gel electrophoresis. We have attempted to find a relationship between the patterns of nuclear proteins and gene expression in mutant plasmacytoma cell lines. The majority of nuclear proteins are common to all of the cell lines studied as would be expected if the majority of nuclear proteins are concerned with functions common to all plasma cells. There are, however, both qualitative and quantitative differences in the nuclear protein patterns of mutant and parent cell lines which appear to correlate with differences in gene expression. The turnover of nuclear proteins in two of the cell lines, MOPC 315.40 (IgA producer) and MOPC 315.32 (lambda 2 chain producer) was studied using pulse-chase techniques.

Quantitation of monoclonal immunoglobulins by immuno-isoelectric focusing and its application for monitoring secretory B cell neoplasia.

A scheme for quantitation of serum and urinary paraproteins is described using isoelectric focusing and scanning densitometry. Paraproteins could be quantified using this system when present at concentrations ranging from 1 mg/ml to 26 mg/ml, depending on the immunoglobulin class. The relevance of these results to monitoring secretory B cell neoplasia and the distinction between monoclonal gammopathy of undetermined significance and myeloma is discussed.

The application of isoelectric focusing to routine screening for paraproteinaemia.

We present a scheme for routine screening of patients' sera for monoclonal immunoglobulin by isoelectric focusing in agarose gels. Using this method combined with immunofixation, we have been able to detect monoclonal immunoglobulin in the sera of patients, and to identify the isotype of the immunoglobulin when conventional techniques such as zonal and immunoelectrophoresis have yielded equivocal results.

Lectin binding to glycoproteins in the surface membrane of Schistosoma mansoni.

A number of lectins were assessed for their ability to bind to glycoproteins in the surface membrane of Schistosoma mansoni. The membrane polypeptides were separated by SDS-PAGE and the glycoproteins visualised by incubating the gel with radio-iodinated lectin followed by autoradiography. Most of the individual lectins bound to a variety of glycoproteins but peanut agglutinin and Dolichos biflorus agglutinin bound preferentially to a single glycoprotein of apparent molecular weight 170 000. This glycoprotein was subsequently shown to be exposed at the surface of the parasite and localised at the tubercles.

Spectrotypes of anti-DNA antibodies show that anti-DNA-secreting B-cell clones of SLE patients are restricted in number, stable and long lived.

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.

Restoration of an early, progressive defect in responsiveness to T-cell activation in lupus mice by exogenous IL-2.

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.

Analysis of the autoimmune response in lupus mice: the behaviour and lifespan of anti-DNA-secreting B-cell clones.

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.

Expression of anti-DNA clonotypes and the role of helper T-lymphocytes during the autoimmune response in mice tolerant to alloantigens.

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.

Serum paraproteins in chronic lymphocytic leukaemia.

The presence of paraproteins in the sera of 10 patients with chronic lymphocytic leukaemia (CLL) was investigated using immunoisoelectric focusing. Monoclonal immunoglobulins were found in nine of these 10 sera. Five sera contained a single monoclonal IgM paraprotein, one serum contained a single monoclonal IgG paraprotein, while three sera contained more than one monoclonal paraprotein--namely, IgM + IgD, IgM + IgG, and IgM + IgD + IgG. The results indicate that the malignant B cells of CLL may be at a later stage of differentiation than previously assumed and serum monoclonal immunoglobulin could be of value as a tumour marker.

Scenarios for autoimmunization of T and B cells in myasthenia gravis.

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.

Naturally occurring antibodies to bovine and human retinal S antigen: a comparison between uveitis patients and healthy volunteers.

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.

A comparison of three methods for production of human hybridomas secreting autoantibodies.

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.