Control of human thyroid autoantibody production in SCID mice.

In order to determine the phenotype of the cells required for thyroid autoantibody production, peripheral blood mononuclear cells (PBMC) from patients with autoimmune thyroid disease (AITD) were transferred to severe combined immunodeficient (SCID) mice. The production of human IgG, thyroglobulin (Tg) antibody and thyroid peroxidase (TPO) antibody in the SCID recipients was monitored for up to 4 months. PBMC from 10 of 13 AITD patients produced substantial IgG (> or = 100 micrograms/ml) and detectable Tg and TPO antibodies in recipient mice. PBMC pretreated to deplete or enrich T cells produced low or undetectable thyroid-specific antibody in SCID mice. Depletion of CD4+ T cells resulted in much lower or undetectable IgG, Tg and TPO antibodies compared with levels seen in recipients of control PBMC. By contrast, depletion of CD8+ T cells from the PBMC had no overall effect on autoantibody production, although with PBMC from some patients CD8+ depletion possibly enhanced both IgG and autoantibody production. In eight of 10 experiments, autoantibody levels reached maximal titres before total IgG levels peaked. It is considered that thyroid autoantibodies are produced from memory B cells activated in SCID mice and that this activation is T cell- and CD4+ T cell-dependent.     

Subpopulations of thyroid autoantibody secreting lymphocytes in Graves'' and Hashimoto thyroid glands.

Lymphocytes isolated from Graves' and Hashimoto thyroid tissue by enzymatic (dispase) digestion or mechanical disaggregation were markedly different in terms of their ability to synthesize thyroid autoantibodies in culture. Dispase digestion, followed by removal of thyroid follicular cells, gave a lymphocyte population with a high T:B cell ratio (6:1). However, the ability of these cell suspensions to synthesize microsomal (Mic) and thyroglobulin (Tg) antibodies spontaneously was significantly increased compared with lymphoid suspensions isolated by mechanical means. Spontaneous synthesis of thyroid autoantibodies was not markedly enhanced in cell suspensions prepared from patients' lymph node tissue by digestion compared with mechanical disaggregation. Further, Mic and Tg antibody production by thyroid lymphocytes prepared using dispase was inhibited by pokeweed mitogen (PWM) whereas in most cases suspensions prepared from the same tissues by mechanical dispersion synthesized low or undetectable levels of autoantibodies whether PWM was present or absent. Digestion of tissue debris remaining after mechanical removal of lymphocytes gave suspensions which had an increased proportion of suppressor/cytotoxic T cells compared with suspensions produced mechanically or by digestion alone; however, in terms of spontaneous autoantibody synthesis and PWM induced inhibition, these suspensions were similar to these obtained by digestion alone. It would therefore seem that enzymatic digestion of thyroid tissue resulted in the isolation of a lymphoid population which was different from that extracted by mechanical disaggregation. The digestion process appears to permit the recovery of lymphocytes closely associated with thyroid follicular cells and our studies suggest that it is this population which makes the major contribution to autoantibody synthesis.     

Prostaglandin-mediated immunoregulation: reduced sensitivity of in vitro immunoglobulin production to indomethacin in rheumatoid arthritis

Spontaneous and pokeweed mitogen (PWM) stimulated in vitro immunoglobulin production from peripheral blood mononuclear cells (PBMC) of control subjects and rheumatoid arthritis (RA) patients both receiving non-steroidal anti-inflammatory drugs (RA + NSAID) was measured by an enzyme linked immunosorbent assay (ELISA). Spontaneous IgG and IgM-RF production from the RA + NSAID was significantly higher than in control subjects. IgM production was also elevated but not significantly. Indomethacin (10(-6) - 10(-8)M) added to in vitro cultures failed to influence spontaneous production from either group. PWM stimulated IgG production was not significantly different between the two groups whilst IgM synthesis was significantly reduced in the RA individuals. IgM-RF production was observed only in the RA + NSAID group. Indomethacin inhibited PWM stimulated IgG and IgM production in control individuals but was significantly less potent on IgG, IgM and IgM-RF production from the RA + NSAID group. This reduction in the inhibitory effect of indomethacin correlated significantly with the high spontaneous immunoglobulin production and a low PWM stimulation index observed in the RA + NSAID group. Indomethacin had no significant effect on PWM stimulated PBMC proliferation in the rheumatoid individuals. These results suggest that B lymphocytes from some RA + NSAID are 'pre-committed' to produce immunoglobulins spontaneously in culture, possibly as a consequence of activation in vivo, and are therefore relatively insensitive to PWM stimulation. These B lymphocytes may have progressed beyond the immunoregulatory steps involving prostaglandins.     

Sites of autoantibody production in rats with thyroiditis.

We have developed a thyroglobulin-specific haemolytic plaque assay and investigated potential sites of autoantibody synthesis in good and poor responder strains of rats immunized with thyroglobulin and in rats subjected to thymectomy and sub-lethal irradiation which subsequently develop thyroiditis spontaneously. The bone marrow appears to be the most important site of thyroglobulin antibody synthesis in all groups, but spleen and cervical lymph nodes are also involved. No thyroglobulin plaque-forming cells could be found in the thyroid. These results imply widespread involvement of the humoral immune system in organ-specific autoimmune processes.     

Cytokines, thyroid autoantibody synthesis and thyroid cell survival in culture

In autoimmune thyroid disease lymphoid cells infiltrating the thyroid gland occur in conspicuous aggregates or as a diffusely distributed population invading the thyroid follicles. Consequently cytokines secreted by activated T cells or macrophages could influence neighbouring thyroid cells as well as other lymphocytes. We have investigated this possibility using recombinant cytokines. Thyroid cell survival was assessed in terms of mitochondrial dehydrogenase activity in monolayers exposed to tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1 (IL-1 alpha and beta) and interleukin-2 (IL-2) in the presence or absence of thyroid-stimulating hormone (TSH). Neither TNF-alpha nor IL-2 affected thyroid cell survival, IFN-gamma was usually inhibitory and IL-1 alpha slightly enhanced cell survival in some experiments. However, the effects were small and variable and were not enhanced by potentially synergistic combinations of cytokines, longer periods of exposure, or different culture conditions. In contrast, IFN-gamma, IL-2 and TNF-alpha inhibited the ability of thyroid lymphocytes from patients with Graves' disease and Hashimoto's thyroiditis to synthesize autoantibodies to thyroid peroxidase (TPO) and thyroglobulin (Tg). Comparison of lymphoid populations isolated by digestion and/or mechanical disaggregation indicated that a population of activated B cells, plasma cells and T cells, intimately associated with thyroid cells since they could only be extracted by digestion, was influenced by cytokines. Our studies suggest that in addition to its well-recognized ability to induce MHC class II antigens on thyroid cells, IFN-gamma may inhibit thyroid cell proliferation and TNF-alpha, IFN-gamma and IL-2 may down-regulate thyroid autoantibody synthesis.     

Mechanism of autoantibody production]

Autoantibody is a hallmark of autoimmune diseases. In systemic autoimmune diseases such as systemic lupus erythematosus, the target of autoantibodies are mostly nuclear autoantigens like nucleosome and U1RNP. Since immune system comprise autoreactivity to develop itself, the chance of autoantibody appearance should not be rare. Therefore, the disturbance of immunoregulation for nuclear autoantigens might allow the persistence of autoantibody production. The studies of tolerance of nuclear antigens is required to understand autoimmune diseases and to develop more advanced immunotherapy.     

In vitro regulation of thyroglobulin (Tg) autoantibody production by Tg-specific T-cell lines and hybridomas.

To define the interactions between self thyroglobulin (Tg)-reactive T and B we co-cultured enriched B cells taken from rat or mouse Tg-primed mice with major histocompatibility complex (MHC) class II-restricted T-cell lines specific for iodinated determinants on self-Tg, or hybridomas derived from those lines. Using two clonally distinct T-cell hybridomas, ADA2 and CH9, in vitro help for Tg autoantibody responses was observed using mouse (M)Tg-primed B cells and a 100 ng/ml MTg challenge. Using rat Tg-primed B cells and the same conditions, only CH9 provided help, indicating that the fine specificity of B cells influences their ability to interact with specific anti-Tg T-cell clones. In contrast to T-cell hybridomas, their parent T-cell lines MTg9B3 and MTg12B suppressed Tg autoantibody responses in vitro, although they augmented bystander proliferation of unprimed B cells. The MTg12B cells also (i) diminished the survival of Tg-primed B cells, and (ii) inhibited the proliferation of an antigen-presenting B-cell hybridoma (LK35.2) in a cytostasis assay. These findings together support the view that their suppressive activity is mediated through cytotoxicity. While the role of class II-restricted cytotoxic cells in thyroid autoimmunity is unknown, the results suggest that such cells may act to suppress autoantibody responses as well as to mediate tissue damage to class II-expressing thyroid cells.     

Production and characterisation of a human monoclonal thyroid peroxidase autoantibody.

A human-mouse hybridoma has been produced by fusion of Hashimoto thyroid lymphocytes with the mouse myeloma line X63-Ag8.653. The cloned hybridoma secreted 2.5 micrograms per 10(6) cells per day of an IgG kappa thyroid peroxidase (TPO) autoantibody (2G4) with high affinity (2.5 x 10(9) molar-1) and specificity for human TPO. 2G4 did not react with lactoperoxidase, horseradish peroxidase or human myeloperoxidase or with porcine TPO or with human thyroglobulin. Plastic tubes coated with 2G4 bound about 50% of 125I-labelled human TPO added and the binding was inhibited by IgGs prepared from 18/18 TPO autoantibody-positive sera. This indicated that all 18 sera contained autoantibodies which recognised the same (or closely related) epitope as 2G4. Plastic tubes coated with IgGs from different TPO autoantibody-positive patient sera also bound 125I-labelled TPO but inhibition by 2G4 in this system was not complete. This suggested that the sera contained at least 2 types of TPO autoantibodies, with only one type of autoantibody reactive with the same epitope as 2G4.     

Studies on toxinogenesis in Vibrio cholerae. II. An vitro test for enterotoxin production

An Elek test for enterotoxin-producing strains of Vibrio cholerae is described. Thirty-five out of 37 strains of classical V. cholerae produced positive reactions, but only 1 of 69 E1 Tor vibrio strains was reactive. Tox(-) mutants of V. cholerae were also unreactive in the Elek test.     

Impaired regulation of erythrocyte autoantibody production after splenectomy.

C3H mice were given 4 i.p. injections, eac of 2 X 10(8) WAG rat RBC, at weekly intervals. The production of erythrocyte autoantibodies elicited by the cross-reacting rat RBC was assessed using the average direct Coombs' test (DCT) score. Autoantibody production reached higher levels and persisted significantly longer in mice splenectomized 15 days before the first injection of rat RBC. This increased production of autoantibodies was not prevented by injecting each splenectomized mouse i.v. with 5 X 10(7) syngeneic spleen cells immediately after splenectomy. Similarly, splenectomy of mice already DCT+ significantly prolonged autoantibody production which was not prevented by injections of 10(8) cells prepared from the spleens removed at splenectomy. Transfer of spleen cells from mice already DCT+ to mice before the injections of rat RBC were started in the recipients caused a significant reduction in the amount of RBC autoantibodies produced. This suppression of autoantibody production was greater in unsplenectomized mice than in splenectomized mice. The results show that the spleen is involved in the regulation of these erythrocyte autoantibody responses. It is hypothesized that, in addition to the cellular component of the spleen, the splenic architecture and/or environment contributes to the regulation of autoantibody responses.     

The genetic basis of autoantibody production

Many autoimmune diseases are characterized by autoantibody subsets that are associated with specific clinical manifestations. The primary genetic associations of these autoantibodies are with MHC genes, most specifically HLA class II, which in many instances better explain the HLA association of the disease per se. It is noteworthy that certain genes and haplotypes, notably HLA-DRB1*0301, DQA1*0501, DQB1*0201 in Caucasians and DRB1*0405, DQA1*03, DQB1*0401 in Asians, as well as PTPN22, seem to be associated with a variety of autoimmune diseases. On the other hand, others are more disease specific (HLA-DRB1*11 for systemic sclerosis and HLA-DRB1 alleles encoding the "shared epitope" in RA) as well as non MHC genes, such as FcyRIIa and IIIa in SLE, the beta2 glycoprotein I gene in the aPL syndrome, and the TSHR gene in Graves' disease). Autoantibody responses also are influenced by the presence of specific MHC and non-MHC genes which may not be associated with the disease per se. These novel associations offer new clues not only to pathogenesis but also to potential therapeutic targets.     

Effect of the xid gene on graft-versus-host-induced autoantibody production in nonautoimmune mice

Graft-versus-host disease (GVH) was used to induce an autoimmune state in F1 recipients using donor spleen cells, splenic T cells, or Lyt 1+2- splenic T cells from either normal DBA/2 mice or from DBA/2 mice carrying the X-linked immunodeficiency (xid) gene. Recipients were either nondefective (DBA/2 X CBA/N)F1 males or reciprocal cross (CBA/N X DBA/2)F1 male mice carrying the xid gene. GVH induced hypergammaglobulinemia and anti-ssDNA autoantibodies in F1 recipients. Immunodeficient (CBA/N X DBA/2)F1 recipients had less hypergammaglobulinemia and IgG anti-ssDNA than did normal (DBA/2 X CBA/N)F1 recipients. Spleen cells, splenic T cells, and Lyt 1+2- splenic T cells from immunodeficient DBA/2.xid donors were less able to induce GVH and autoimmunity than normal DBA/2 donors. These studies suggest that the xid gene may reduce the autoimmune hyperractive state, but may do so by acting on more than one cell population, including T cells.     

Effect of the BXSB Y chromosome accelerating gene on autoantibody production

The Y chromosome of the BXSB mouse is able to accelerate and adversely alter the autoimmune disease of inbred BXSB mice and in male F1 hybrids. In order to further study the effects of the BXSB Y chromosome, we developed three inbred congenic strains, each bearing the BXSB Y: CBA/J.BXSB-Y, NZW.BXSB-Y, and NZB.BXSB-Y. The BXSB Y did not induce anti-DNA or anti-red blood cell (RBC) autoantibodies in either CBA/J or NZW congenic strains. Thus, it is an accelerating rather than an inducing factor. NZB.BXSB-Y congenic mice had accelerated anti-RBC but not anti-DNA. Studies of recombinant inbred by BXSB F1 mice indicated that the BXSB Y did not act to promote the activity of the NZB gene underlying anti-DNA. These and studies of (BXSB X NZB.BXSB-Y) F1 mice suggested that BXSB autosomal genes are required for the full anti-DNA accelerating activity of the BXSB Y. These mice provide a basis for future molecular genetic studies of the BXSB Y.     

Epidermal acantholysis induced in vitro by pemphigus autoantibody. An ultrastructural study.

Suprabasilar acantholysis can be produced in organ culture of normal human skin in the presence of pemphigus IgG autoantibody. We have examined this in vitro system by electron microscopy. The earliest ultrastructural changes at 12 hours included widening of the intercellular spaces and disruption of the intercellular cement substance in the nondesmosomal areas. After 24 to 48 hours in culture, the tonofilaments retracted from the cell periphery, desmosomes were lost, and extensive cell surface digitation occurred. By 72 hours, isolated cells without noticeable desmosomes were seen in the suprabasilar areas, whereas basal cells, with intact hemidesmosomes, remained attached to the basal lamina. Control cultures which were grown in the presence of normal IgG or F-10 medium alone did not manifest these changes. The ultrastructural features support the conclusion that the acantholysis produced in this system is similar and probably identical to that of naturally occurring pemphigus.     

胰岛素样生长因子1的免疫调节作用的体外实验研究

目的：探讨胰岛素样生长因子１（ＩＧＦ１） 的免疫调节作用。方法：随机抽取健康个体８２ 名和肿瘤个体１２６ 名,体外提取外周血淋巴细胞后将ＩＬ２ 和或ＩＧＦ１ 共同培养,用３ ＨＴｄＲ 掺入法、３ ＨＴｄＲ 释放法及流式细胞仪检测淋巴细胞增殖活性、杀伤活性和ＣＤ４＋ ＣＤ８＋ 比率。结果：适当浓度的ＩＧＦ１（５０ ｎｇｍｌ）在体外能协同ＩＬ２ 促进ＬＡＫ细胞的增殖,增强其杀伤Ｒａｊｉ细胞的活性,提高淋巴细胞亚群ＣＤ４＋ ＣＤ８＋ 的比率。结论：ＩＧＦ１ 在体外对人体外周血免疫细胞起正向调节作用。