Humoral and cell-mediated immunity in experimental progressive thyroiditis in rabbits.

Rabbits immunized over a long period of time with serial injections of aqueous preparations of either bovine thyroglobulin or chemically altered rabbit thyroglobulin develop progressive thyroiditis. As is short-term thyroiditis in rabbits and mice, this thyroiditis is characterized by lesions and cellular infiltration similar to that observed in Arthus reactions. Once the progressive thyroiditis is established, the rabbits respond readily to subsequent injections of native rabbit thyroglobulin. No significant reduction of lesions or circulating antibody is observed when injections of native rabbit thyroglobulin are substituted for the preparations used to induce the disease. Cell-mediated hypersensitivity to rabbit thyroglobulin, as evidenced by MIF activity, develops in rabbits after prolonged immunization with altered or cross-reacting thyroglobulin. It is suggested that this activity develops as a result of a loss in the unresponsive state in T lymphocytes. The data indicate that it is the persistence of circulating antibody to autologous thyroglobulin which sequesters autologous thyroglobulin from peripheral lymphoid tissue, and thus, results in the loss of the unresponsive state in lymphocytes of these tissues. It is suggested that similar events may be involved in the development of cell-mediated hypersensitivity in thyroiditis in humans.     

Cell-mediated responses to heterologous and homologous thyroglobulin in guinea-pigs immunized with heterologous thyroid extracts

Immune cellular responses and circulating antibodies to heterologous and homologous thyroglobulin have been studied in two groups of guinea-pigs immunized with human or bovine thyroid extract in Freund's complete adjuvant. In animals immunized with human thyroid extract, the in vitro [2-¹⁴C]thymidine incorporation by lymphocytes and the inhibition of peritoneal exudate cell migration in the presence of human thyroglobulin were earlier and more marked than those to bovine thyroglobulin as observed in animals immunized with bovine thyroid extract. In the two groups of guinea-pigs no significant difference was found regarding the production of circulating antibodies.

Moreover cellular cross-reaction to homologous thyroglobulin could be detected in animals immunized with human but not in those immunized with bovine thyroid extract. Serological and cellular cross-reactions between human and bovine thyroglobulin were present in both groups of guinea-pigs.

Finally a significantly higher incidence of thyroid inflammatory lesions was found in the human thyroid extract immunized animals. The role of cell-mediated immune responses in initiating tissue damage in experimental thyroiditis is discussed.

     

Perpetuation of autoimmune thyroiditis and production of secondary renal lesions following periodic injections of aqueous preparations of altered thyroglobulin

Periodic injections of aqueous preparations of thyroglobulin coupled to the diazonium derivatives of arsanilic and sulphanilic acids (arsanil–sulphanil thyroglobulin) resulted in perpetuation of both the synthesis of circulating antibody to native thyroglobulin and thyroiditis. Two months after the last injection, the rabbits made a strong immune response to a subsequent injection of native thyroglobulin. Without the periodic injections both circulating antibody and thyroid lesions disappeared and the ability to respond to native thyroglobulin was lost. The level of circulating antibody and the incidence and severity of lesions were considerably greater in rabbits receiving periodic injections over a 6-month period of time than in rabbits given a series of injections during a period of 1 month. The relation of these findings to the progressive nature of some autoimmune diseases is discussed. Complex-induced renal injury, secondary to the immune response to arsanil–sulphanil thyroglobulin, was observed in the rabbits given the periodic injections over a 6-month period of time. Chronic glomerulonephritis apparently resulted from deposition of thyroglobulin–anti-thyroglobulin complexes and complement along the glomerular basement membranes.     

Comparative effects of immunization of rabbits with human thyroglobulin and human and rabbit thyroid microsomal fractions

Dutch rabbits were immunized with human thyroglobulin and human and rabbit thyroid microsomal fractions. The animals were bled at intervals and their serum was assayed for thyroid-stimulating activity, thyroglobulin haemagglutinins, and total and free thyroxine (T₄). Their thyroidal radio-iodine uptake and thyroid histology was also studied. Five out of ten animals immunized with human thyroglobulin developed histological evidence of thyroiditis but none had thyroid-stimulating activity in the serum. Only one out of twenty-six animals immunized with rabbit or human thyroid microsomal fractions had any histological abnormality in the thyroid, but eight had significant amounts of thyroid-stimulating activity in their serum. Although the frequency of the latter response was no greater after immunization with rabbit as compared to human thyroid microsomal fraction, there was a significant increase in serum free T₄ in the group immunized with rabbit tissue.

Thyroiditis and rabbit immunologic thyroid stimulator (RITS) formation appear to be completely separable phenomena, and RITS is not a by-product of thyroiditis. The absence of thyroid-stimulating activity in the serum of rabbits immunized with thyroglobulin is further evidence that thyroglobulin is not the important antigen leading to the production of this thyroid-stimulating globulin.

     

Immune complex disease. VII. Experimental mesangiopathic glomerulonephritis produced by chronic immunization with thyroglobulin.

Immunohistologic and electron microscipic studies were performed on the kidneys of rabbits given daily intravenous injections of porcine thyroglobulin in amounts adjusted to the immune response of the individual rabbits. Glomerular lesions were restricted to the mesangium, were characterized by varying degrees of proliferation of mesangial cells and increase of mesangial matrix, and were accompanied by accumulations of rabbit immunoglobulins, C3, and porcine thyroglobulin. Electron-dense deposits were localized to the mesangium and the adjacent subendothelial space. Less than 10 per cent of the animals with mesangila lesions developed obvious impairment of glomerular function. Thyroglobulin-containing immune complexes were found to be rapidly removed from the mesangium, so that overloading of the mesangium and consequent accumulation of complexes in the adjacent capillary loops could not occur. Thus, the results provide further evidence that when immune complex deposition is restricted to the mesangium, relatively little interference with glomerular function results. This situation is paralleled in man by the lesions of subclinical lupus nephritis, chance proteinuria and hematuria, and the early lesions of Berger's disease.     

Immune responses to thyroglobulin in experimental allergic thyroiditis

Guinea-pigs immunized with homologous thyroglobulin in complete Freund's adjuvant were observed for lesions in the thyroid gland, and for development of specific delayed type skin hypersensitivity. Their blood leucocytes were examined for specific sensitivity in vitro by determining the inhibition of migration in the presence of thyroglobulin. Serum was tested for humoral antibodies by the passive haemagglutination technique. There was a significant correlation between the development of thyroiditis and the intensity of the skin reactions. No relationship was observed between the presence of thyroiditis and leucocyte sensitivity nor between thyroiditis and circulating antibodies. On the other hand serum antibody titres were correlated to the degree of leucocyte sensitivity. Thyroglobulin inhibited the migration of cells from normal animals in the presence of plasma from thyroglobulin-sensitized animals. The activity of sensitized plasma disappeared when diluted 1:10 in normal plasma irrespective of the serum antibody titre. Guinea-pigs immunized with thyroglobulin or kidney antigen in complete Freund's adjuvant and tested for skin or leucocyte hypersensitivity showed specific but no cross reactivity to the two preparations.     

Suppression of thyroid lesions in rabbits by treatment with cyclophosphamide after the induction of thyroiditis

An acquired immunologic unresponsive state to bovine thyroglobulin has been induced in adult rabbits following simultaneous injections of cyclophosphamide and bovine thyroglobulin. The acquired unresponsive state in some rabbits lasted at least 3 months after the last injection of bovine thyroglobulin. After induction of autoimmune thyroiditis by soluble injections of altered homologous arsanilsulphanil thyroglobulin or bovine thyroglobulin, a decrease in the incidence of thyroid lesions and the level of antithyroglobulin antibody production was observed following simultaneous injections of cyclophosphamide and native thyroglobulin.     

Thyroid peroxidase and the induction of autoimmune thyroid disease

Animal models of autoimmune thyroid disease are associated with thyroglobulin (Tg) as autoantigen whereas in man the autoimmune response to microsomal antigen/thyroid peroxidase (TPO) appears to play a major role in thyroiditis. Consequently, we have compared the ability of TPO and Tg to induce thyroid autoantibodies and thyroid damage in mice known to be susceptible (CBA/J) or resistant (BALB/c) to thyroiditis induced using murine Tg. Groups of three to five mice were immunized twice using Freund's complete adjuvant with 80-100 micrograms highly purified porcine (p) TPO, pTg, rat (r) Tg, human Tg, bovine serum albumin (BSA) or BSA + 0.2 micrograms pTg (the level of Tg contamination of TPO). Four weeks after immunization with TPO, plasma from CBA/J (but not BALB/c) mice contained IgG class antibodies which bound to TPO-coated tubes in the presence or absence of excess Tg (and could therefore be clearly distinguished from Tg antibodies) but there was no evidence of thyroiditis in either strain of mice. In contrast, in CBA/J mice immunized with rTg and, to a lesser extent in mice that had received pTg, thyroid tissue was infiltrated with lymphoid cells and/or neutrophils and antibodies to pTg (but not pTPO) were present. Our observations demonstrate that induction of TPO antibody alone is insufficient to lead to thyroiditis in CBA/J mice. Further, these studies emphasize the complex interactions between MHC and different thyroid antigens in the processes leading to thyroid destruction.     

Effect of the synthetic immunomodulator, linomide, on experimental models of thyroiditis

The drug Linomide is an immunomodulator showing marked down-regulation of several experimental autoimmune diseases. In this study, its effect on three different experimental models of thyroid disease and on spontaneous infiltration of salivary glands (sialoadenitis), was investigated. Although very effective at preventing thyroid infiltrates in mice immunized with mouse thyroglobulin and complete Freund's adjuvant and in spontaneous models of thyroiditis and sialoadenitis, it completely failed to modify experimental autoimmune thyroiditis (EAT) induced in mice immunized with mouse thyroglobulin and lipopolysaccharide. There was no significant shift in the observed isotypes of anti-mouse thyroglobulin antibodies and only anti-mouse thyroglobulin antibodies in the spontaneous model were completely down-modulated by the drug. One surprising fact to emerge was that Linomide-treated donor mice, although protected from thyroid lesions themselves, were still able to transfer EAT showing that they must have been effectively primed while being treated with Linomide. It is possible that the drug down modulated EAT by interfering with the trafficking of primed effector cells.     

Effect of the Traditional Korean Immunomodulating Formulation,Gamguntang (GGT), on Experimental Thyroiditis Model

The crude herbal formulation, Gamgungtang (GGT), is an immunomodulator showing marked down-regulation of several experimental autoimmune diseases. In this study, its effect on different experimental models of thyroid disease was investigated. Although very effective at preventing thyroid infiltrates in mice immunized with mouse deglycosylated thyroglobulin and complete Freund's adjuvant and in spontaneous models of thyroiditis, it completely failed to modify experimental autoimmune thyroiditis (EAT) induced in mice immunized with mouse thyroglobulin and lipopolysaccharide. There was no significant shift in the observed isotypes of anti-mouse thyroglobulin antibodies and only anti-mouse thyroglobulin antibodies in the spontaneous model were completely down-modulated by the GGT. One surprising fact to emerge was that GGT-treated donor mice, although protected from thyroid lesions themselves, were still able to transfer EAT showing that they must have been effectively primed while being treated with GGT. It is possible that the drug down modulated EAT by interfering with the trafficking of primed effector cells.     

Effect of the traditional Korean immunomodulating formulation, Gamguntang (GGT), on experimental thyroiditis model

The crude herbal formulation, Gamgungtang (GGT), is an immunomodulator showing marked down-regulation of several experimental autoimmune diseases. In this study, its effect on different experimental models of thyroid disease was investigated. Although very effective at preventing thyroid infiltrates in mice immunized with mouse deglycosylated thyroglobulin and complete Freund's adjuvant and in spontaneous models of thyroiditis, it completely failed to modify experimental autoimmune thyroiditis (EAT) induced in mice immunized with mouse thyroglobulin and lipopolysaccharide. There was no significant shift in the observed isotypes of anti-mouse thyroglobulin antibodies and only anti-mouse thyroglobulin antibodies in the spontaneous model were completely down-modulated by the GGT. One surprising fact to emerge was that GGT-treated donor mice, although protected from thyroid lesions themselves, were still able to transfer EAT showing that they must have been effectively primed while being treated with GGT. It is possible that the drug down modulated EAT by interfering with the trafficking of primed effector cells.     

Thyroglobulin induced lymphocytic thyroiditis in two sublines of BB/Wor rats.

The BB/Wor rat develops spontaneous insulin dependent diabetes mellitus (DM) and lymphocytic thyroiditis (LT). We have recently demonstrated that although the incidence of spontaneous DM is relatively constant among different inbred BB/Wor sublines the incidence of LT is extremely variable. Experimental LT can be induced in some animal species by immunization with thyroglobulin (Tg). The differences in susceptibility of Tg induced LT between a high incidence LT subline (NB) and a low incidence subline (BB) were determined after immunization with Tg obtained from Wistar rat thyroids. Immunization was accomplished using 0.6 mg Tg in complete Freund's adjuvant (FA) or FA alone at 30 and 37 days. Since spontaneous LT rarely occurs before age 75 days, rats were sacrificed at age 65 days to specifically study Tg induced LT. Immunization with Tg induced LT in the NB subline but not in the BB subline. Anti-Tg antibody (Ab) titers, T4-Ab and T3-Ab were all increased in both Tg immunized sublines but were significantly higher in Tg immunized NB rats than in Tg immunized BB rats. The increase in T4-Ab or T3-Ab resulted in factitiously low serum T4 and T3 values when a single Ab technique with polyethylene glycol (PEG) precipitation was used in the RIA. There was a dissociation in the incidence of Tg induced LT and Ab production. Although Tg immunization failed to induce LT in the BB subline, anti-Tg Ab were significantly elevated as well as both T4-Ab and T3-Ab, suggesting that anti-Tg Ab titers per se are not tightly correlated with the occurrence of LT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Splitting of human thyroglobulin. II. Enzymatic digestion

The antigenic mosaic of human thyroglobulin was studied following proteolysis by papain, pepsin, trypsin and chymotrypsin. Undigested thyroglobulin (Tg) was removed from the enzyme-derived fragments by gel filtration. The fragments produced by the action of trypsin precipitated with rabbit heteroantibody to thyroglobulin but failed to precipitate when tested with human autoantibody to thyroglobulin. They only weakly inhibited the tanned cell haemagglutination reaction using either human or rabbit antiserum and failed to elicit antibody following injection with Freund's adjuvant into rabbits.

The fragments produced by the proteolytic action of pepsin and papain differed from the trypsin product in that they precipitated with anti-normal human thyroid extract rabbit serum and (in the case of the pepsin fragment) human thyroiditis serum, and strongly inhibited the tanned cell haemagglutination reaction of either antiserum. They also elicited thyroglobulin-specific antisera in rabbits.

Chymotrypsin-produced fragments failed to precipitate with, or to inhibit, either rabbit or human antisera. They were not immunogenic in rabbits.

     

Characterization of different types of experimental autoimmune thyroiditis in the Buffalo strain rat

We have compared features of experimental thyroiditis in the Buffalo strain rat induced by neonatal thymectomy, immunization with rat thyroglobulin (Tg) and complete Freund's adjuvant, and subcutaneous administration of trypan blue or 3-methylcholanthrene. The disease produced by neonatal thymectomy resulted in significantly worse thyroiditis and higher antibody levels than the other models and shared other features in common with Hashimoto's thyroiditis. In particular TSH levels were elevated, Tg antibodies had a restricted subclass distribution, the thyroid was infiltrated by both B cells and T cells (comprising equal numbers of W3/25 and Ox8 positive cells) and thyroid follicular cells were Ia antigen-positive in some of the thymectomy group animals. In the other forms of thyroiditis, the thyroid infiltrate was mainly composed of macrophages or dendritic cells and B cells. Different pathogenic mechanisms are probably involved in these models; the disease produced by neonatal thymectomy shows the closest similarity to Hashimoto's thyroiditis.     

Inhibition of the production of autoimmune thyroiditis in the rabbit

Thyroiditis and antibody to native thyroglobulin developed in rabbits given two series of injections of an aqueous preparation of homologous thyroglobulin, coupled to the diazonium derivatives of arsanilic and sulphanilic acids. The production of both thyroid lesions and antibody were inhibited when native homologous thyroglobulin was injected either prior to, or simultaneously with, the injections of the arsanil—sulphanil-thyroglobulin. The autoimmune response induced following injections of arsanil—sulphanil-thyroglobulin was re-stimulated by a single injection of native thyroglobulin given at a later time; however, multiple injections failed to re-stimulate and the lesions disappeared. The ability to produce autoimmune thyroiditis by injections of aqueous preparations of arsanil—sulphanil-thyroglobulin appeared to be highly dependent on the injection schedule and/or the injection dose.     

Autoimmune murine thyroiditis. VIII. Role of different thyroid antigens in the induction of experimental autoimmune thyroiditis.

Mice of the C57Br strain, which are susceptible to the induction of autoimmune thyroiditis with mouse thyroglobulin, and C57Bl mice, which are resistant, were immunized with human and rabbit thyroglobulins in Freund's complete adjuvant. Susceptible strain C57Br developed higher degrees of thyroid infiltration than the resistant strain. The results indicate that the responses to xenogeneic (foreign) thyroglobulins parallel allogeneic and syngeneic (mouse) thyroglobulin. BSVS mice, which are highly susceptible to thyroiditis, were immunized with mouse thyroid extract from five different mouse strains including syngeneic antigen. Recipients of C57Bl and DBA thyroid extracts showed lower indices of pathology than recipients of similar extracts from C3H, BSVS and non-inbred CF-1 mice. The results suggest that there is a difference in the immunogenicity of mouse thyroid extracts from different strains. Purified thyroglobulin was prepared from congenic strains B10.D2 (H-2d, resistant) and B10Br (H-2k, susceptible). H-2k thyroglobulin gave a greater response in both H-2k and H-2d mice than H-2d thyroglobulin.     

Immunological events leading to destructive thyroiditis in the AUG rat.

Single or infrequent observations in patients or animals with autoimmune thyroid disease (AITD) have failed to elucidate the exact sequence of pathogenetic events leading to thyroid cell destruction. A detailed serial morphological and functional study of experimental AITD (EAITD) in the female AUG rat was therefore undertaken. Following induction of EAITD with thyroglobulin (Tg) in adjuvant antibodies to Tg were detectable one week after the initial immunization, at which stage Ia positive vascular endothelium was observed within the thyroid. This was followed by large numbers of Ia positive dendritic-like cells. With time, in almost all the animals whose titre of Tg antibody rose above a critical level, lymphocytic infiltration was observed consisting mainly of Ia positive B cell aggregates with fewer scattered T cells. This was associated with raised levels of serum TSH and concomitant focal follicular hyperplasia and necrosis. Expression of Ia was mainly restricted to the outer epithelial wall of follicular thyrocytes in direct contact with invading lymphoid cells, although occasional staining on the internal apical membrane was observed as a late event in the destructive process. The Ia expression on thyroid epithelial cells was only observed in areas of thyroid lymphoid infiltration. The immune infiltration of the thyroid in the AUG rat appears to be very similar to that observed in Hashimoto's thyroiditis, with the exception that Ia was not regularly observed on the apical surfaces of thyrocytes. Whether or not the diminished or absent epithelial Ia expression contributes to the spontaneous recovery of the disease observed in this model remains to be resolved.     

Prevention of autoimmune thyroiditis in T cell-depleted rats by injections of crude thyroid extract.

The effect of intraperitoneal injections of crude allogeneic thyroid extract on the development of autoimmune thyroiditis in PVG/c rats subjected to thymectomy and repeated sublethal irradiation has been examined. Treatment with rat thyroid extract during the course of irradiation and before the onset of thyroiditis prevented the expected development of thyroid lesions and autoantibodies to thyroglobulin in these animals. Rats treated with crude rat liver extract under the same conditions developed thyroid disease similar to that of untreated controls. Treatment of thymectomized and irradiated rats with thyroid extract after the completion of the irradiation schedule failed to reduce the incidence and severity of thyroiditis in these animals as compared to that of untreated controls. The possible significance of these findings in relation to the pathogenesis of autoimmune thyroiditis in T cell-depleted rats is discussed.     

Target organ susceptibility and autoantibody production in an animal model of spontaneous autoimmune thyroiditis.

F1-hybrids of Obese strain (OS) chickens, afflicted with spontaneous autoimmune thyroiditis (SAT), and normal, inbred CB chickens, do not develop severe thyroiditis. About 50% of these crosses show circulating autoantibodies to thyroglobulin (TgAAb), but the thyroid glands are only slightly infiltrated, suggesting that the target organ is not susceptible to autoimmune attack. In the present study we show that despite this mild infiltration TgAAb are only synthesized by lymphoid cells within the thyroid gland. Furthermore, we demonstrate that immunization with chicken thyroglobulin (Tg) in complete Freund's adjuvant causes severe experimental autoimmune thyroiditis (EAT) in F1(OSxCB) hybrids.     

Localized primary amyloid tumor of the thyroid developing in the course of Hashimoto's thyroiditis.

A case of localized primary amyloid tumor of the thyroid gland developing in the course of Hashimoto's thyroiditis was studied using histochemistry, immunohistochemistry and electron microscopy. The patient was diagnosed as having Hashimoto's thyroiditis by histological examination of the thyroid and by the presence of a high titer of serum thyroglobulin and thyroid microsomal antibodies. In addition, the thyroid gland exhibited multiple nodular deposits of amyloid which were resistant to prior incubation with potassium permanganate. The amyloid deposits were surrounded by numerous histiocytes and multinucleated giant cells which contained small amyloid droplets in their cytoplasm. However, no amyloid deposits were observed in the walls of blood vessels. Immunohistochemistry showed that the amyloid was strongly positive for amyloid P component, IgG and kappa light chains. Ultrastructurally, the amyloid was composed of straight fibrils with a diameter of 7 to 10 nm. Histiocytes extended slender cytoplasmic processes in a radial fashion into amyloid fibrils, which exhibited a highly organized star-like pattern. This was considered to be an extremely rare case of localized primary amyloidosis of the thyroid, in which IgG, especially kappa light chains (AL), was present as a precursor protein.