Ultrastructural study of spontaneous bone marrow rosette-forming cells.

Mouse bone marrow contains spontaneous rosette-forming cells (RFC) which include more than 70% T-cell precursors, as assessed by their transformation into theta-positive cells after incubation with thymic hormone. Such spontaneous RFC, examined in C57B1/6 mouse bone marrow by electron and scanning electron microscopy, have consistently been shown to be small, inactive mouse lymphocytes when macrophages have been eliminated by cell preincubation. These data suggest that thymic hormone target cells include small quiescent lymphocytes.

Covalent cross-linking of thyrotropin to its receptor on cloned hybrid human thyroid cells (GEJ).

Purification of the TSH binding sites from cloned human thyroid hybrid cells (GEJ) was performed after biosynthetic labeling of the cells, and affinity chromatography on a human TSH-Sepharose column and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The relative Mr of the GEJ cell TSH receptor (TSH-R) was found to be approximately 48,000. This was confirmed by cross-linking [125I]TSH to GEJ binding sites with two homobifunctional agents: dimethyl suberimidate and disuccinimidyl suberate. We found that the cross-linked complexes had a Mr of 78,000 thus yielding a TSH-R size of 48 kilodaltons, after subtraction of the 30 kilodalton TSH, based upon the cross-linking of one TSH molecule per binding site. Moreover, the absence of a dithiothreitol effect demonstrated that the TSH binding site on GEJ cells was formed by a single chain lacking disulfide bonds. Finally trypsinization of TSH/TSH-R complexes detected two tryptic sites yielding two fragments with respective Mr of approximately 2,000 and 15,000.

Characterization of monoclonal antibodies to the human thyrotropin receptor.

We have produced four monoclonal antibodies (mAbs), 34A, 49G, 11E7, and 12E3, which bind the human TSH receptor (hTSH-R) when expressed on a human thyroid cell line (GEJ), freshly dissociated human and murine thyroid cells, or Chinese hamster ovary cells stably transfected with the hTSH-R gene. These mAbs were obtained after immunization of DBA/1 mice with affinity-purified TSH-binding sites from GEJ cells. Biochemical studies, including sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, Western blot, and immunoprecipitation of solubilized GEJ cell membranes or human thyroid cells showed that most of the mAbs recognized two bands: one located at 46-48 kilodaltons and the other at 86-88 kilodaltons. Inhibition of [125I]hTSH binding to solubilized porcine membranes (TSH-receptor auto-antikörper assay) or Chinese hamster ovary cell membranes previously transfected with hTSH-R gene showed that mAb 34A recognizes the hTSH-binding site of both receptors. In contrast, mAbs 49G, 11E7, and 12E3 recognize a structure located near the hTSH-binding site. Lastly, the ability of these mAbs to stimulate murine thyroid function was investigated by measuring cAMP production and iodide accumulation. The 34A mAb, which fully competes with [125I]TSH for binding to hTSH-R, was able to induce both functions. Conversely, the 12E3 mAb, which was the least potent inhibitor of [125I]TSH binding to hTSH-R-transfected cells had no effect. A relationship was, therefore, established between the capacity of mAb to hTSH-R to inhibit [125I]hTSH binding and their ability to induce thyroid functions.

Normal suppressive T cell function of Epstein-Barr virus-induced B cell activation in Graves' disease.

Several studies have demonstrated abnormalities of T cell regulation of Epstein-Barr virus (EBV)-induced B cell activation in systemic autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and systemic sclerosis. To investigate whether this abnormality is a common feature of other autoimmune diseases, we studied 10 EBV-immune normal subjects and 22 EBV-immune patients with Graves' disease (GD); 11 had newly diagnosed hyperthyroidism, and 11 had received carbimazole treatment for hyperthyroidism for at least 6 months. Peripheral B lymphocytes infected with EBV were cultured for 20 days in the presence or absence of autologous T cells at different ratios. Immunoglobulins M and G secretion into the supernatants was determined using an enzyme-linked immunosorbent assay. The extent of suppression when T cells were added, as measured by a suppression ratio, was not significantly different in normal subjects and newly diagnosed GD patients (0.65 vs. 0.63 on the 16th day and 0.77 vs. 0.72 on the 20th day of culture, respectively). In carbimazole-treated patients, the appearance of functional suppressor T cells was delayed slightly, but the overall suppression ratios on the 16th and 20th days were normal. Thus, a T cell regulation abnormality of EBV-induced B cell activation could not be demonstrated in patients with untreated hyperthyroid GD, suggesting that the autoimmune reactivity in such patients is probably dependent upon a specific thyroid suppression defect rather than a generalized suppression defect.

Production and characterisation of monoclonal antibodies directed against different epitopes of human thyroid stimulating hormone.

We have produced monoclonal antibodies (mAbs) to human thyroid stimulating hormone (hTSH) and selected five that specifically recognize hTSH and do not cross-react with the other human glycoprotein hormones such as luteinizing hormone (LH), chorionic gonadotropin (CG), follicle stimulating hormone (FSH). All of the antibodies were of the IgG1 subclass with affinities ranging from 5.3 X 10(8) to 1.9 X 10(10) mol-1.l; they could be assigned to two subgroups on the basis of their epitope specificity.

Induction of Ia antigens on thyroid cultures by syngeneic T lymphocytes: a model of autoimmune disease.

We previously reported that the expression of Ia antigens on cultured monolayers of murine thyroid epithelial cells (TEC) occurred with a particular distribution exclusively on the basal part of the cultured thyroid cells, while class I antigens of the major histocompatibility complex (MHC) are only detected on the apical surface. It appears that deposition of syngeneic lymphocytes induces, 24 h later, Ia expression on the apical side of cultured TEC, the surface that is in direct contact with the responder lymphocytes during syngeneic sensitization of T lymphocytes. We hypothesized that this phenomenon could represent, in syngeneic situations, the restriction process in antigen recognition by T cells, as demonstrated by Ia restricted primary syngeneic sensitization (PSS) on murine TEC.

Lymphoid cells from the rodent Praomys (Mastomys) natalensis express antigenic determinants present on mouse and rat lymphocytes.

Recent studies have suggested that Praomys (Mastomys) natalensis might be a suitable model for analyzing the underlying mechanisms of autoimmunity, since a high incidence of autoantibodies to a wide variety of specificities occurs in this rodent which is intermediate in size between the mouse and the rat. Mastomys, also called the "multimammate mouse" or "multimammate rat", more closely resembles the rat than the mouse as regards some anatomical traits, and differs from both common rodents as to others. Because of controversies many years ago, Mastomys was never phylogenetically classified as a genus, but remains a subgenus, designated Praomys (Mastomys) natalensis. We have attempted to determine whether immunological markers differentiated on mouse or rat lymphocytes are present on Mastomys lymphoid cells. It was demonstrated that Mastomys cells (thymus, spleen, lymph node, bone marrow) bear theta determinants of both mouse and rat, express mouse membrane Ig, and bind with anti rat Ia antigens (which cross-react with mouse specificities). Functional markers (helper or suppressor/cytotoxic functions of the mouse or rat) were never found on Mastomys lymphoid cells.

Human autologous rosette-forming cells. V. Study of MHC control in erythrocyte-lymphocyte interaction.

The role of the major histocompatibility complex (MHC) in human autorosette formation was studied. On a large series of healthy subjects who were typed for HLA antigens, we tested in blind rosette formation with 90 autologous and 295 allogeneic red blood cells (RBC). We found that the mean levels of auto- and allorosettes were similar, being significantly higher in females than in males. However, we failed to find any role for blood group antigens and any involvement of HLA antigens in the interactions between lymphocytes and RBC in rosette formation. Moreover, high or low autorosette levels were not associated with a particular HLA allele. The comparison of individual percentages of auto- and allorosettes indicated that 51% of the subjects displayed identical levels of auto- and allorosettes whereas 29% formed preferentially rosettes with auto- rather than allo-RBC and 20% exhibited lower autorosette than allorosette levels. Among the group of subjects who were better responders for autorosettes than allorosettes, we found an increased frequency of the haplotype A29, B44. Taken together these findings suggest that in contrast to the murine situation, the autorosetting phenomenon in humans is not HLA restricted.

T cell mapping of one epitope from thyroglobulin inducing experimental autoimmune thyroiditis (EAT).

These data collect the advance made in the last few years in our laboratory in defining one epitope from the thyroglobulin (Tg) molecule (660 KDa) inducing Experimental Autoimmune thyroiditis (EAT) in CBA/J mice. We achieved the characterization of one EAT-inducer Tg peptide by combining "in vitro" biochemical and immunological approaches and "in vivo" studies. Since T cells recognize degraded forms of the antigen and since endogenous antigens preferentially activate class I-restricted T cells, we hypothesized that one cytotoxic T cell hybridoma, named HTC2, which prevents further EAT induction in mice injected with Tg would be specific for one EAT inducer peptide. In order to identify one Tg epitope inducing EAT, enzymatic treatment of the protein by trypsin, HPLC purification and sequence analysis were performed. Simultaneously, tryptic digests were used to pulse CBA/J macrophages and tested for their ability to be recognized by HTC2 cells. Lastly, when digests were recognized by HTC2 cells their capacity to induce EAT in CBA/J mice was evaluated. To further assess the pathogenicity of the sequenced Tg peptide, one synthetic peptide was made and its capacity to induce EAT verified. By this procedure we identified for the first time one 40 amino-acid peptide from human thyroglobulin inducing EAT in CBA/J mice.

Normal suppressive T cell function of Epstein-Barr virus induced B cell activation in type 1 (insulin dependent) diabetes mellitus.

Several studies have demonstrated abnormalities of T cell regulation of Epstein-Barr virus-induced B cell activation in systemic autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematous, and systemic sclerosis. However, a normal suppressive peripheral T cell function was observed in Graves' disease. To investigate whether this abnormality is a common feature to other autoimmune diseases, we studied T cell regulation of Epstein-Barr virus induced B cell activation in 15 newly diagnosed type 1 (insulin dependent) diabetes mellitus patients and 10 normal control subjects. Peripheral B lymphocytes infected with Epstein-Barr virus were cultured for 20 days in the presence or absence of autologous T cells at different ratios (1:1 and 1:4). IgM and IgG secretions into the supernatants were determined using an enzyme-linked immunosorbent assay. The extent of suppression when T cells were added, as measured by a suppression ratio, was not significantly different in type 1 (insulin dependent) diabetes mellitus patients and normal subjects. We conclude that in type 1 (insulin dependent) diabetes mellitus, the autoimmune reactivity is not dependent upon a generalized suppression defect. It can be hypothesized, therefore, that in type 1 diabetes mellitus as well as in Graves' disease, a local or organ specific suppressor deficit may induce the autoimmune phenomena.

Ig VH gene family usage in spleen cells of CBA/J mice immunized with experimental autoimmune thyroiditis (EAT) inducer antigens.

Experimental autoimmune thyroiditis (EAT) is an autoimmune disorder of the thyroid gland induced in susceptible strains of mice by thyroglobulin (Tg). We recently showed that low Mr (< 10 kDa) Tg tryptic fragments and a 40 amino-acid peptide (F40D) from Tg could induce EAT as well as native Tg. Because it has been reported that autoantibodies (A-Abs) express VH families preferentially located in the D-proximal VH gene segment, we investigated whether A-Abs specific for one pathogenic peptide from Tg were also skewed towards D proximal VH gene segment. In that respect, we immunized CBA/J mice with EAT inducer antigens of decreasing sizes: Tg (660 M(r)), < 10 kDa Tg trypic fragments or F40D peptide (4.9 kDa M(r)) from Tg. The VH gene segments utilized by immune spleen cells were determined by hybridization to total spleen cell RNA previously deposited onto nylon membranes and densitometric scans. This study was conducted on days 7 and 9 after determination of the maximum amounts of mRNA coding for immunoglobulins and on day 28 when A-Ab levels are the highest. Results were compared to VH gene segment expression both in normal and adjuvant-injected mice. We found that immunization of CBA/J mice with EAT inducer antigens stimulate B cells the restriction of which, in terms of VH family usage, depends on the size of the immunizing antigen: the larger the antigen, the higher the numbers of VH families used. Moreover, we found that B cell stimulation consecutive to immunization with the peptidic antigen inducing EAT occurs in VH Q52 family, a VH encoded by D-proximal gene segment.

Desensitization of the antigen receptor primes B cells for activation by superstimulatory influenza virus.

Interaction of the B cell receptor (BCR) with non-immunogenic receptor ligands can induce a specific state of B cell unresponsiveness as a result of receptor desensitization. Provided it is maintained over time, BCR desensitization may provide the molecular basis for clonal anergy. Using an in vitro model of anti-Ig-mediated BCR desensitization, we assessed the susceptibility of desensitized "anergic" B lymphocytes to activation by B cell superstimulatory influenza virus hemagglutinin (HA). Rabbit anti-mouse Ig antibodies (whole Ig molecule or F(ab')(2)-fragments) totally abolished the response of murine B cells to HA, when added simultaneously with the virus. Pretreatment with the same antibodies, however, yielding a complete unresponsiveness to a subsequent challenge with normally mitogenic anti-Ig reagents, even enhanced the subsequent proliferative response to HA. By contrast, HA-mediated high-rate immunoglobulin synthesis was suppressed after desensitization. BCR-desensitized, HA-stimulated B cells exhibited a hyperexpression of various activation markers (B7, major histocompatibility complex class II, CD25) and served as potent antigen-presenting cells (APC) in a polyclonal model for T lymphocyte activation. These observations suggest a possible scenario for the breaking of natural B cell tolerance, where infections with B cell superstimulatory viruses may lead to the clonal expansion of receptor desensitized, functionally silenced B lymphocytes in vivo.

Effects of murine recombinant interleukin-10 on the inflammatory disease of rats transgenic for HLA-B27 and human beta 2-microglobulin.

Rats transgenic for HLA-B27 and human beta 2-microglobulin develop a spontaneous, multisystem, inflammatory disease that resembles human B27-associated disease and that involves the gut mucosa. This model predominantly affects the colon and is characterized by an extensive infiltration of the mucosa by inflammatory cells, largely composed of mononuclear cells. In addition, an increased plasma level of nitric oxide (NO)-derived metabolites was described in this model. Deficiency in the anti-inflammatory cytokine, interleukin-10 (IL-10), leads to the development of colitis in IL-10 knockout mice, suggesting that IL-10 plays a major role in the control of gut inflammation. The objectives of this work were to study the mechanisms of the inflammatory bowel disease (IBD) in HLA-B27 rats and to determine the effects of treatment with IL-10. The 33-3 line of HLA-B27 recombinant rats with established disease was treated in two consecutive experiments with murine recombinant IL-10 for five weeks. Assessment of the effect of this treatment was performed, based on clinical, histological and biological (myeloperoxidase and inducible NO synthase activities; tumor necrosis factor-alpha, interferon-delta, CD3, iNOS and beta-actin mRNA expression. In 33-3 rats with established disease, mesenteric lymph nodes were hyperplastic, and colonic cellularity and MPO and iNOS activities in the colonic mucosa were increased without any detectable effects of IL-10 administration. IFN-gamma and iNOS mRNA were only detected in the colon of transgenic rats. Despite a lack of effect on disease expression, IL-10 strikingly reduced the level of IFN-gamma mRNA in gut mucosa. Up-regulation of IFN-gamma mRNA suggests that the IBD of HLA-B27 rats is mediated by T-helper 1 lymphocytes. Sustained administration of IL-10, in HLA-B27 rats with established disease, efficiently inhibited IFN-gamma mRNA expression but did not influence disease expression: these results indicate that IFN-gamma may exert a critical role at an earlier stage of the disease rather in the maintenance of the lesions.

Induction of experimental autoimmune thyroiditis (EAT) by heat-denaturated thyroglobulin (Tg).

Experimental Autoimmune Thyroiditis (EAT) is characterized by autoreactive T and B cell responses, assessed by a marked lymphocytic infiltration of the thyroid gland by T cells and the occurrence of circulating autoantibodies (AAb) to thyroglobulin (Tg). It was recently reported that administration of denaturated exogenous antigens primes class I-restricted cytotoxic T cells in vivo. Since cytotoxic T cells are involved in EAT development, porcine Tg (pTg) was heat-denaturated, i.v. injected into CBA/J mice and features of EAT evaluated. Simultaneously, mice were immunized with pTg and adjuvants and evaluation of EAT performed. We found that heat-denaturated pTg (hdpTg) induced EAT in recipient mice similar to native pTg/adjuvants. Surprisingly, whereas Tg-specific cytotoxic T cells were regularly found in lymph node cells from hdpTg or native pTg immunized mice, proliferative responses were only detected using T cells from native pTg immunized mice. Autoantibodies to pTg were decreased by a factor 30 in sera from mice immunized with hdpTg. These data further emphasized the role of Tg-specific cytotoxic T cells in EAT.

[Study of thyroid autoimmunity in man. Contribution of cell cultures to the study of antibodies stimulating the thyroid gland in Basedow's disease]. / Exploration chez l'homme de l'auto-immunité thyroïdienne. Apport des cultures cellulaires à l'étude des anticorps stimulant la thyroïde dans la maladie de Basedow.

The value of thyroid cell cultures in vitro has been demonstrated in auto-immune thyroiditis in animals. We have used the same method in man to investigate for serum thyroid-stimulating antibodies (TSAb) by measuring cyclic AMP production in thyroid cell cultures incubated with immunoglobulins from patients with Graves' disease. The specificity of the reaction is strictly directed either against TSH or against immunoglobulins from patients with Graves' disease; 92% of the sera of our untreated Graves' disease patients were positive. The value of this technique compared to the other methods used for detecting TSAb is discussed.

[Predisposition to thyroid autoimmune diseases]. / Prédisposition aux maladies autoimmunes thyroïdiennes.

OBJECTIVES: Genetic predisposition is required for the expression of thyroid autoimmune disorder addition to the immune dysfunction and the environmental factors. METHODS: In order to evaluate the role of this genetic factor, we reported the results of immunological and hormonal investigations of 62 members (TD), belonging to a large Akr family, who are related to 40 patients with Graves' disease or Hashimoto's thyroiditis. RESULTS: The hormonal analyses showed that 19 subjects exhibited an infraclinical hypothyroidism, subdivided into 7 members with pathological rates of TSH evocative of thyroid insufficiency and 12 others with compensative thyroid insufficiency. Seventeen subjects of the Akr family who had solely antithyroid autoantibodies were considered as potential candidates to develop thyroid autoimmune diseases. The clinical follow-up, during two years, confirmed the diagnosis of Hashimoto's thyroiditis in 3 members among 19 subjects with infraclinical hypothyroidism (TD05, TD28 and TD54) and in only 1 member out of the 17 potential candidates (TD03). CONCLUSION: Our results showed that a serological study of hormones and/or autoantibodies directed against thyroid antigens, could allow the detection of predisposed subjects to develop a thyroid autoimmune pathology. The Akr family seems to be suitable for the study of the localization of susceptibility genes to TAID.