Influence of diet on the induction of experimental autoimmune thyroid disease.

Immunization of CBA/J mice with thryoglobulin (Tg) emulsified in complete Freund's adjuvant induces experimental thyroiditis (EAT), a well-characterized model of Hashimoto's disease. Recent studies have suggested that dietary factors play a role in the modulation of the immune response and that diet can have a profound effect on the induction of autoimmune diseases. In this study, we examined the influence of diet on autoimmune thyroiditis in mice. EAT was induced in mice fed ad libitum one of the three diets, a standard maintenance chow (Agway H1000), Purina 5020 Breeding Chow, and Purina 5010 Autoclavable (unautoclaved) Diet. Tg-immunized mice fed the Agway 1000 diet were found to be resistant to the development of autoimmune thyroid disease, with only 4 out of 25 mice developing mild thyroiditis. In contrast, 16 out of 25 mice fed the Purina 5010 diet developed moderate to severe thyroiditis. Mice fed the 5020 diet were partly susceptible: 7 out of 25 developed a mild to moderate thyroiditis. Histologic examination of thyroid glands of diseased mice fed the 5010 and 5020 diets showed marked lymphocytic infiltration with destruction of follicles, compared with mice fed the Agway diet, the latter showing only mild infiltration with preservation of thyroid follicles. Titers of antibody to Tg did not differ among the groups, and there was no significant difference in the IgG isotype subclass usage. The results demonstrate that diet can markedly affect the severity of autoimmune disease in the EAT model. In contrast, diet has little effect on the humoral autoimmune response in this system. These results implicate diet as a factor in the severity of cell-mediated autoimmune destruction and suggest that dietary modification could decrease pathology in some forms of autoimmune disease.

Mouse thyroglobulin: conservation of sequence homology in C-terminal immunogenic regions of thyroglobulin.

cDNA encoding 287 amino acids of the C-terminus of mouse thyroglobulin was cloned and sequenced. The amino acid homology between mouse and rat thyroglobulin was 96%, and was 78% between mouse and human. It was found that mouse thyroglobulin completely shared homology with two thyroiditogenic peptides described by other investigators. These findings are consistent with our hypothesis that in murine experimental thyroiditis, the primary thyroiditogenic epitopes are encoded by mouse-specific regions of thyroglobulin.

Silicone binding immunoglobulins in human sera.

We have evaluated two recently published test procedures for demonstrating antibodies to silicone. Positive control serum samples provided to us by the original authors were positive in our hands, using the methods described. However, serum samples from patients with certain connective tissue diseases but no history of silicone implants were also positive. The question of silicone-specific antibodies remains unresolved.

IgG subclass distribution of thyroglobulin antibodies in patients with thyroid disease.

The IgG subclass distribution of thyroglobulin antibodies (TgAb) has been studied in Hashimoto and Graves' patients by several investigators with conflicting results, in part explainable by methodological problems. We have recently developed a quantitative ELISA to measure in absolute terms the serum concentration of TgAb subclasses. The aim of the present study was to apply this method in a large series of patients with autoimmune as well as, for the first time, non-autoimmune thyroid diseases. We examined 28 patients with Hashimoto's thyroiditis, 30 with Graves' disease, 21 with thyroid carcinoma and 18 with non-toxic goitre, all selected for the presence of TgAbs. The results indicated that TgAbs in thyroid diseases were not restricted to any particular isotype, but comprised all four IgG subclasses. IgG1 was represented similarly in the four groups. The same was true for IgG3, even though its contribution to the total antibody content was very small. IgG4 was the dominant subclass in patients with Graves' disease, thyroid carcinoma and non-toxic goitre, probably reflecting a prolonged antigenic challenge. In Hashimoto's thyroiditis IgG2 was dominant, possibly because T helper lymphocytes infiltrating the thyroid are typically Th1 type.

Tryptic peptides of human thyroglobulin: I. Immunoreactivity with murine monoclonal antibodies.

Human thyroglobulin (Tg) was treated with trypsin at different concentrations of trypsin/Tg for various incubation times at 37 degrees C using non-reducing conditions. A ratio of trypsin to Tg of 1:100 (w/w) was optimal to release small peptides that were reactive to murine MoAbs to human Tg. Most peptides were released after only 1 h incubation with trypsin, but these peptides were further degraded at longer incubation times. However, a few small peptides, the largest of which with an apparent molecular weight (MWap) of 40 kD, resisted tryptic digestion up to at least 12 h of incubation. These resistant peptides were further degraded by trypsin at 18-24 h of incubation. Tryptic peptides of Tg, released at 1 h and 4 h of incubation, were analysed for their immunoreactivity to 16 well characterized anti-Tg MoAbs by Western immunoblot. Patterns of peptide recognition of these MoAbs were generally unique. Eight MoAbs reacted with peptides of MWap of 10-25 kD and above. Four other MoAbs reacted with peptides of MWap of 25-43 kD and above, and the remaining four reacted with peptides of MWap > 43 kD. Nine of these MoAbs failed to recognize peptides after reduction, suggesting that the MoAbs bind conformation-dependent epitopes. The above information will promote the development of models relating the structure of Tg to the autoimmune process, and may provide an understanding of those regions of Tg responsible for the induction of autoimmune thyroiditis.

Immunoreactivity of multiple molecular forms of human thyroglobulin.

Human thyroglobulin (Tg) was purified from thyroids of normal individuals and of patients with Graves' disease using gel filtration (Sephacryl S-400) and ion-exchange (DEAE) column chromatography. We isolated five protein peaks of Tg from the DEAE column, using a step gradient, characterized them for protein and iodine content, and assessed their immunological properties by reactivity to polyclonal and monoclonal antibodies (mAbs). Normal and Graves' Tgs differed in the relative protein content of these five protein peaks from the DEAE column. In the case of normal Tg, the majority of Tg was eluted in peak 2 but in the Graves' Tg, most of the protein was eluted in peak 1 of this column. The immunoreactivity of these five protein peaks of Tg was studied using 11 mouse mAbs prepared against human Tg, sera from patients with autoimmune thyroiditis and polyclonal antibody from a rabbit immunized with human Tg. All of five protein peaks of Tg reacted equally with rabbit antibody. The sera of five thyroiditis patients showed greater binding to peak 1 of Graves' Tg than peak 1 of normal Tg. Similarly, most mAbs showed greater binding to peak 1 of Graves' Tg than the peak 1 of normal Tg. Of particular interest was one mAb (42C3) which reacted only with Tgs containing iodine. The immunoreactivity of this mAb paralleled the iodine content of Tg. This mAb might be useful for evaluating the role of iodine in the antigenicity of human Tg.

Effects of early and late treatment with anti-CD4 monoclonal antibody on autoimmune disease in MRL/MP-lpr/lpr mice.

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop a systemic autoimmune disease, characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation, with target organ inflammatory lesions composed largely of CD4+ (helper) T cells. Previous reports have demonstrated that anti-CD4 monoclonal antibody (mAb) treatment of MRL/lpr mice from 1 to 5 months of age resulted in a dramatic reduction in both the frequency and the severity of autoimmune disease. In order to investigate the effects of early, short-course and late, short-course anti-CD4 mAb therapy on the autoimmune disease in MRL/lpr mice, groups of 12 to 15 animals were treated with weekly intraperitoneal injections according to one of four regimens: (i) anti-CD4 mAb from age 1 to 5 months (continuous treatment); (ii) anti-CD4 mAb from age 1 to 3 months (early treatment); (iii) anti-CD4 mAb from age 3 to 5 months (late treatment); and (iv) either normal saline or rat immunoglobulin (control treatment). Continuous treatment resulted in a dramatic reduction of both frequency and severity of the autoimmune disease, as demonstrated histologically and serologically. Early treatment also resulted in a significant reduction in autoimmune disease, while late treatment had little effect. Glomerulonephritis was detected in none of the animals in the continuously treated group (P < 0.05), 38% of those in the early-treated group (P = < 0.05), 92% of the late-treated group, and 100% of controls. The titer of antinuclear antibodies, of anti-dsDNA antibodies, and total immunoglobulin levels were all significantly reduced in the continuous-treatment and early-treatment groups, but not in the late-treatment group. Murine antibodies to rat anti-CD4 mAb were present in the late-treatment group. These results indicate that early short-course anti-CD4 mAb treatment of MRL/lpr mice is effective in ameliorating the autoimmune disease in this model, while late-treatment is ineffective, probably due to the induction of antibody directed against anti-CD4 mAb itself.

Epitopes on thyroglobulin: a study of patients with thyroid disease.

Thyroglobulin antibodies (TgAbs) are typically found in autoimmune thyroid diseases and, more rarely, in nonautoimmune thyroid diseases and healthy subjects. To determine whether TgAbs associated with different conditions recognize different epitopes on the thyroglobulin molecule, we studied 28 patients with Hashimoto's thyroiditis, 30 with Graves' disease, 21 with thyroid carcinoma, 18 with nontoxic goiter, and 25 healthy subjects. All patients were selected for the presence of TgAbs; 4/25 healthy subjects also had TgAbs. The sera were assayed for the their ability to inhibit the binding of monoclonal antibodies to thyroglobulin in an ELISA assay. We found that: 1) TgAbs in Hashimoto's patients preferentially recognized three clusters of epitopes (II, III and typically VI), with no difference between the goitrous and the atrophic variants; 2) TgAbs in Graves' patients were directed toward cluster II, with no difference between the presence or the absence of ophthalmopathy; 3) TgAbs in thyroid carcinoma patients recognized the same clusters as Hashimoto's patients; 4) TgAbs in nontoxic goiter patients and in the four healthy subjects showed no restriction in epitope recognition. We suggest that in individuals with no overt clinical or biochemical thyroid abnormalities but with TgAbs, the finding that these TgAbs recognize particular immunodominant clusters may be utilized to predict full-blown thyroid disorders. Longitudinal studies are needed to evaluate the possible clinical application of this methodology.

The IgG2a antibody response to thyroglobulin is linked to the Igh locus in mouse.

The IgG-subclass usage by several strains of mice in the response to immunization with mouse thyroglobulin (mTg) was examined in the experimental autoimmune thyroiditis model. While the subclass usage by most mouse strains was similar, the Ighb allotype-bearing mice consistently produced lower IgG2a levels to mTg. Using CBA-Ighb congenic and recombinant inbred strains of mice, the lower level of IgG2a in the Ighb mouse was mapped to the Igh locus. The regulation of IgG2a appeared to be cis controlled, as the CBA x C57BL/6F1 mouse also produced reduced IgG2a of the Ighb (B6) allotype but not of the Ighj (CBA) allotype.

Quantitative measurement of human thyroglobulin-specific antibodies by use of a sensitive enzyme-linked immunoassay.

A quantitative enzyme-linked immunoassay that measures in absolute terms the subclass concentration of human thyroglobulin (huTg)-specific IgG autoantibody was developed. Unique to this study was the use of an affinity-purified anti-huTg standard with a known concentration of the four IgG subclasses. The sensitivity of the ELISA assay was 1-5 ng/ml depending on the IgG subclass being measured. We examined 22 sera of patients with autoimmune thyroid disease. The total huTg-specific antibody concentrations in serum ranged from 0 to nearly 3000 micrograms/ml of IgG. The IgG subclass distribution in individuals with low huTg-specific IgG (< 10 micrograms/ml) was primarily IgG1 and IgG3 Ab. Patients with intermediate levels of huTg IgG (10-600 micrograms/ml) expressed all four subclasses; however, no particular subclass was dominant. Individuals with > 1000 micrograms/ml also showed huTg-Ab in all four subclasses, however, IgG1 and IgG2 were dominant. All four IgG subclasses were used in the response to huTg, although the pattern of usage varied between individuals. There was no dominant subclass usage seen in this patient population.

Anti-CD4 monoclonal antibody therapy suppresses autoimmune disease in MRL/Mp-lpr/lpr mice.

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop systemic autoimmune disease, characterized by vasculitis, lymphadenopathy, glomerulonephritis, and autoantibody formation. The target organ inflammatory lesions are composed largely of CD4+ "helper" T cells, while the massively enlarged lymph nodes are composed primarily of CD3+ CD4- CD8- TCR alpha/beta + "double-negative" T cells. In this study we investigated the effect of treatment of MRL/lpr mice with anti-CD4 monoclonal antibody (mAb); control groups consisted of animals treated with normal saline or rat immunoglobulin (Ig). Anti-CD4 mAb treatment, which was started at 4 weeks and continued through 20 weeks of age, resulted in a dramatic reduction of both the frequency and severity of the autoimmune disease, as demonstrated histologically and serologically. Anti-CD4 mAb therapy markedly reduced the frequency of glomerulonephritis and eliminated vasculitis of the major renal arterial branches. Glomerulonephritis was detected in 9 of 9 saline-treated, 9 of 9 rat Ig-treated, but in only 1 of 9 anti-CD4 mAb-treated mice; vasculitis was detected in 6 of 9 saline-treated, 7 of 9 rat Ig-treated, but in none of 9 anti-CD4 mAb-treated mice. The frequency of antinuclear antibodies, titer of anti-dsDNA antibodies, and total Ig levels were all significantly reduced by anti-CD4 mAb therapy. These data support the hypothesis that CD4+ T cells play a central role in the disease process in this autoimmune strain.

The frequency of LPS-responsive B cells to autologous and heterologous thyroglobulin.

The frequency of precursors within the mouse splenic B cell pool, reactive with mouse thyroglobulin (mTg) was estimated using a limiting dilution assay system. The mean frequency was found to be 1/3900 B cells. The results provide a minimal estimate of the frequency of mTg-reactive B cells. The frequency of mTg-reactive B cells was not influenced by the MHC locus, as both high- and low-responder strains showed similar frequencies. While the frequency of B cells reactive to human Tg was found to be similar to that reactive to mTg, only 20% of the mTg-reactive clones also cross-react with human Tg. Similarly, only 30% of huTg reactive clones were found to react with mTg. Therefore, a large proportion of Tg-reactive antibodies are restricted to self-determinants and not determinants to conserved regions of the Tg molecule.

Mapping the autoepitopes of thyroglobulin.

Chronic thyroiditis is currently the best available model of an organ-restricted autoimmune disease, because the principle antigen Tg is a well-characterized protein and the disease can be reproduced by experimental immunization. The pattern of autoantigenic determinants of Tg that is recognized by sera from subjects without detectable thyroid disease is clearly different from that seen with thyroiditis patients. Both sets of sera react with the evolutionary conserved portions, the hormonogenic regions containing the T3 and T4 moieties. Sera of thyroiditis patients, however, also react with the evolutionary variable, species-specific portions of the Tg molecule. Mice immunized with human Tg respond to the same, immunodominant sites. Mice injected with mouse Tg preferentially recognize the analogous, mouse-specific sites of Tg. Mice are not restricted, however, in their use of V-(D)-J genes for Ig production, even when the same antigenic determinant is involved.

Autoantibodies to thyroglobulin are encoded by diverse V-gene segments and recognize restricted epitopes.

Murine experimental autoimmune thyroiditis has been used as a model for human autoimmune thyroiditis. Experimental autoimmune thyroiditis is induced in mice by immunization with mouse thyroglobulin (Tg) in CFA. To characterize the antibodies to this autoantigen, we have studied the binding specificities and determined the nucleotide sequences of monoclonal anti-Tg antibodies. The specificities of the mAb for determinants on Tg varied extensively. Seven of 16 mAb showed reactivity to only mTg, 4 reacted to Tg from more than one species and four reacted to a variety of Ag. Many of the mAb were competitively inhibited by thyroid hormones, suggesting that they recognize the hormonogenic sites on the Tg molecule. The mAb could be divided into at least seven reactivity patterns based on reciprocal competitive inhibition studies, indicating that mTg contains at least seven antigenic regions. DNA sequence analysis of the mAb showed that a large number of V region gene segments encoded the H and L chains. No evidence for preferential use of any V region family or gene segment was found. Gene segments from the VH 7183, Q52, J558, and VH10 families were used by heavy chains, and the V kappa 1, 4, 8, 9, 19, and 21 families were used by kappa-chains. The results indicate that the antigenic epitopes on mTg elicit a very diverse autoantibody response that is derived from a large number of V region gene segments. Many of these autoantibodies show specific reactivity with mTg indicating they recognize species specific epitopes. The results suggest that clonal deletion of autoreactive Ab to certain self-epitopes may not occur.

Cloning and characterization of a cDNA that encodes a 70-kDa novel human thyroid autoantigen.

cDNA clones were isolated by screening a human thyroid carcinoma lambda gt11 library with immunoglobulins purified from serum of a patient with autoimmune Graves' disease. One clone (ML8) containing a 1.25-kilobase (kb) insert hybridized with a single 2.0-kb poly(A+) mRNA in human thyroid and lymphocytes but not in human brain, liver, kidney, or muscle. In addition, this probe also hybridized with a single 2.0-kb poly(A+) mRNA from a rat thyroid cell line (FRTL-5). An apparently full length 2,074-base pair (bp) human cDNA was obtained and sequenced. The nucleotide sequence of the 2,074-bp cDNA includes a 5'-noncoding sequence of 17 bp, a 1827-bp open reading frame, and a 222-bp 3'-noncoding sequence. The canonical polyadenylation signal AATAAA is present 18 bp upstream of the poly(A) tail. This cDNA encodes a 69,812-dalton protein with two potential N-linked glycosylation sites and at least one potential membrane spanning domain. Immunoprecipitation of the in vitro translated protein by sera from several patients with Graves' disease argues that the 69,812-dalton protein is an autoantigen.

The induction of organ-specific antibodies during the graft-vs.-host reaction.

During the parent (P) into F1 hybrid graft-vs.-host reaction (GVHR), nuclear, leukocyte and erythrocyte autoantibodies are commonly seen. The specificity of these autoantibodies is reminiscent of those found in systemic lupus erythematosus (SLE) patients and SLE-prone mice. Organ-specific antibodies, however, including thyro-globulin (Tg) antibodies do not arise spontaneously. There have been conflicting reports about the ability of exogenous Tg to induce an anti-Tg response during the GVHR. We have re-examined this question in greater detail. Using the murine P----F1 GVHR system, the results of this work demonstrate that mouse thyroglobulin (MTg)-specific antibodies can be induced during a GVHR. However, mice must both be undergoing a GVHR, and have received exogenous MTg. The highest autoantibody response occurs if mice are injected with mouse thyroid extract or purified MTg at the time of P----F1 cell transfer. The anti-MTg response is MTg dose dependent. The ability to induce anti-MTg antibody was not major histocompatibility complex restricted, for both the DBA/2----B6D2F1 (low responder H-2 haplotypes to MTg), and AKR or DBA/2----AKD2F1 (high/low responder----high responder haplotype) GVHR gave similar responses. The anti-MTg titers peaked between days 7-10 and declined thereafter. In contrast, antibodies to dsDNA were not present at this early time, but developed after several weeks. We conclude that organ-specific autoantibodies can be induced during a GVHR if the appropriate antigen(s) are presented near the time of GVHR induction.

The growth of concanavalin-A activated, Lyt selected subsets in IL-2 containing supernatants.

The growth properties of Con A activated, Lyt selected splenic T lymphocytes were examined by limiting dilution analysis and clonally by single cell picking. Under the conditions used, a comparable frequency of Lyt 1+ and Lyt 2+ cells grew after Con A activation in the presence of Con A rat spleen supernatant. At a clonal level, however, the growth of these subsets differed qualitatively and quantitatively. While Lyt 2+ cells obtained clone sizes of several hundred cells, Lyt 1+ clone sizes were usually less than 100 cells, and many clones aborted their growth after a few days. Morphologically, the Lyt 1+ cell was smaller and usually showed fewer pseudopodial protusions as compared to the Lyt 2+ cell.

Quantitative studies on T-cell functions in MRL/MP-Lpr/Lpr mice. I. Frequency analysis of precursor cells of proliferating, cytotoxic, and T-cell growth factor-secreting T lymphocytes reveals an increase in absolute numbers, with a concomitant decrease in percentage of immunocompetent cells.

A limiting dilution system has been applied to compare precursor frequencies of proliferating T lymphocytes (PTL-P), of T-cell growth factor-secreting T cells (TTCGF-P), and of cytotoxic T cells (CTL-P) in lymphocyte populations of aged, MRL/MP-lpr/lpr (MRL-lpr) mice and the congeneic strain MRL/MP- +/+ (MRL-n,), or the H-2k-compatible strains AKR/N and B10.BR responding to the mitogen concanavalin (ConA), to alloantigens (H-2) or to trinitrophenol (TNP)-modified syngeneic cells. In lymph node and spleen populations of 3- to 8-month-old MRL-lpr mice, the frequencies of H-2d- or ConA-reactive PTL-P and TTCGF-P, and of CTL-P sensitive to either H-2d, TNP, or ConA stimuli were between 5 to 30 times lower than in the corresponding populations of the other three strains. Furthermore, the frequencies of CTL-P progressively decreased in MRL-lpr mice from 3 to 8 months of age. In contrast, the absolute numbers of immunologically competent precursor T cells (PTL-P, TTCGF-P, CTL-P) was in general approximately 2- to 5-fold higher in MRL-lpr than in the control mice. However, these normal T cells do not seem to expand proportionally with the progressive lymphadenopathy in MRL-lpr mice since the number of T lymphocytes recovered from lymph nodes of the individual animals tested exceeded those of tissues from control mice by 30- to 300-fold. The results therefore suggest that mature T cells are progressively diluted out by abnormal lymphocytes in lymphocyte populations of aging MRL-lpr mice, thus causing a decrease of immune responses in vitro and possibly also affecting optimal cellular interactions in vivo.