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.

     

Relationship between the tuberculin-type and Jones-Mote-type hypersensitivities: suppression of basophil infiltration by mycobacterial adjuvant

Guinea-pigs immunized with bovine gammaglobulin (BGG) in incomplete Freund's adjuvant (IFA) showed the typical Jones-Mote-type hypersensitivity (JMH) reaction when tested 5 days later. This is characterized by prominent basophil infiltration. After pretreatment with complete Freund's adjuvant (CFA) 16 days before immunization with BGG in IFA, the lesions resembled the JMH reaction macroscopically in their evolution over time and in the absence of a positive macrophage migration inhibition (MIT) test. However, histologically, the lesions resembled classical tuberculin-type hypersensitivity with prominent mononuclear cell infiltration without any basophils. The pretreated animals, which failed to show basophil infiltration, were able to transfer JMH reactions with basophil infiltration into normal animals. In contrast, pretreatment of recipients with CFA or Corynebacterium parvum prevented the passive transfer of the characteristic effect on the JMH reaction when given shortly before skin testing. We postulate that macrophages activated by CFA may play an important role in regulating basophil infiltration in the effector phase of the delayed hypersensitivity reaction.     

Delayed Hypersensitivity, Macrophage Migration, and Antibodies in Guinea-Pigs Immunized with Diphtheria Toxoid

Guinea-pigs were immunized with diphtheria toxoid (DT) in Freund's complete adjuvant (FCA) and boosted three times with DT in saline. Boosting increased anti-DT passive haemagglutinins, haemolysins, and cytophilic antibody on the surface of peritoneal exudate cells, but skin reactions to DT became negative and peritoneal cell migration was no longer inhibited by DT. When normal peritoneal cells were incubated in the sera of boosted animals, their migration was not inhibited by antigen, although they had cytophilic antibody on their surface as shown by rosette formation.     

Starch dermatitis: evidence of immunogenicity of surgical glove powder in the guinea-pig.

Guinea-pigs immunized by nuchal inoculation of starch glove powder emulsified with Freund's complete adjuvant showed increased thickening of the ear erythema and monoculear inflammation after local injection of 0.1 mg starch compared with similarly treated control animals inoculated with saline and Freund's complete adjuvant. The responses in the ear of the immunized animals were most pronounceed 3-4 weeks after immunization and 24-72 h after local challenge. It is suggested that the inflammatory response in the immunized guinea-pig has 2 components: reaction to a primary irritant in the glove powder and delayed hypersensitivity to an unidentified component of the glove powder.     

Delayed Hypersensitivity, Macrophage Migration, and Antibodies in Guinea-Pigs Immunized with Diphtheria Toxoid

Guinea-pigs were immunized with diphtheria toxoid (DT) or with DT-anti-toxin precipitate, both in Freund's complete adjuvant (FCA). DT-induced inhibition of peritoneal cell migration was equally strong in both groups and increased with time after immunization. Some guinea-pigs immunized with DT-antitoxin precipitate were boosted with DT or DT-antitoxin precipitate, both in FCA. This increased the antibody titre but did not affect 24-hour skin reactivity or migration inhibition. Migration inhibition did not correlate with anti-DT passive haemagglutinins, haemolysins, or cytophilic antibody on peritoneal cells, but did correlate with 24-hour skin reactivity.     

Adjuvant properties of Micropolyspora faeni.

The adjuvant properties of Micropolyspora faeni, an important source of antigenic material in the production of farmer's lung, were evaluated by comparing antibody- and cell-mediated immune responses of rabbits to bovine serum albumin (BSA) incorporated in complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and incomplete Freund's adjuvant with 5-10 mg/ml homogenized M. faeni (MFA). Rabbits immunized with BSA in CFA or MFA developed significantly increased antigen-induced macrophage migration inhibition, lymphocyte stimulation, and delayed skin reactivity when compared to those immunized with BSA in IFA. No similar adjuvant effect on specific antibody production was observed in rabbits immunized using BSA in MFA. These data suggest that M. faeni can act as a selective immunologic adjuvant for delayed hypersensitivity. This adjuvant property might be important in the induction of mononuclear cell infiltrates seen in human hypersensitivity pneumonitis.     

Immunoadjuvant properties of glove cornstarch powder in latex-induced hypersensitivity

BACKGROUND: Cornstarch powder present in medical gloves plays an important role in latex-induced hypersensitivity as allergen carrier either, by the inhalation route, by skin contact or by direct contact with mucous membranes. OBJECTIVE: Our objective was to test the hypothesis that cornstarch could act as an immunoadjuvant in immediate type-I latex-induced hypersensitivity. METHODS: Guinea-pigs were sensitized by intraperitoneal route with two different antigens (latex proteins and ovalbumin) with or without cornstarch powder. Airway responsiveness after specific bronchial provocation was evaluated and specific IgG and IgG1 levels were determined by enzyme-linked immunosorbent assay (ELISA). Controls were treated with cornstarch powder or saline alone. RESULTS: Animals sensitized with latex proteins (n = 7 in each group) showed significant bronchoconstriction (P < 0.03) and higher anti-latex antibody levels than the controls (P < 0.005). Guinea-pigs sensitized with latex-contaminated cornstarch had higher levels of specific antibodies than those sensitized with latex alone (P < 0.05). Animals sensitized to latex mixed with cornstarch showed higher bronchospasm than those treated with latex alone (P < 0.003). Animals sensitized to ovalbumin mixed with cornstarch also showed higher antibody and bronchoconstriction levels (P < 0.05) than those immunized with ovalbumin alone but antibody titres were significantly lower than those of the animals treated with ovalbumin and Freund's complete adjuvant (P < 0.01; n = 5 in each group). CONCLUSION: Our findings show that cornstarch powder increases antigen-induced bronchoconstriction and antibody production. This role of immunoadjuvant is not antigen-specific. The cornstarch powder used as donning agent in latex gloves is an allergen carrier and it can enhance latex-induced hypersensitivity.     

In vitro transfer of cellular immunity in experimental allergic orchitis by means of immune RNA.

Ribonucleic acid (RNA) extracts were obtained from lymph nodes of guinea-pigs that had previously been immunized with a purified testicular antigen emulsified in Freund's complete adjuvant. The RNA extracts were incubated with normal guinea-pig peritoneal exudate cells (NGP-PEC). After treatment, the NGP-PEC cells showed specific inhibition of migration when tested with the specific antigen. No inhibition of migration was observed when cells were tested with an unrelated antigen or when the cells were incubated with RNA obtained from animals immunized with adjuvant alone. Failure of inhibition of migration was also observed when the 'immune' RNA was degraded with RNAse. The appearance of this I-RNA in the immunized guinea-pigs correlates with the appearance of delayed hypersensitivity in vivo.     

Inhibition of delayed hypersensitivity by pre-immunization without complete adjuvant

Guinea-pigs were immunized by injections of blood group substance with incomplete adjuvant, followed after an interval of approximately 2 weeks, by intracutaneous immunization with the same antigen and Freund's adjuvant containing M. tuberculosis. This treatment inhibited the appearance of delayed skin reactions, while circulating antibody production took place as in controls which had received complete adjuvant only with blood group substance, and had delayed skin reactions. The inhibition of the skin reaction was found to be antigen-specific with regard to unrelated antigens, but showed cross-inhibition for serologically different human blood group substances. The first immunization had to be given more than 2 days before the immunization with complete adjuvant. A similar phenomenon was seen with ovalbumin as antigen. In addition to inhibition of the delayed skin reaction, there appeared to be less γ₂-antibody to ovalbumin than in ovalbumin plus complete adjuvant-only controls. Passive administration of antibody did not affect the development of a delayed hypersensitivity state in complete adjuvant-immunized animals with blood group substance or ovalbumin as antigen. Present evidence favours an explanation of the phenomenon in terms of temporary paralysis on the part of some of the antibody-producing cells—viz. those concerned with delayed hypersensitivity and γ₂-antibody production.     

Immune deviation: demonstration of split tolerance in vitro by inhibition of macrophage migration

Migration of cells, taken from animals immunized with ovalbumin in Freund's complete adjuvant which gave normal delayed hypersensitivity skin responses, was found to be significantly inhibited in the presence of antigen. Migration of peritoneal exudate cells from guinea-pigs in which immune deviation had been induced by immunization with antigen in Freund's incomplete adjuvant was not inhibited.     

The suppression of experimental thyroiditis in the rat by heterologous anti-lymphocyte globulin

The auto-allergic thyroiditis produced in rats by the injection of rat thyroglobulin in complete Freund's adjuvant can be suppressed by the administration of the IgG immunoglobulin fraction of heterologous (horse) anti-lymphocytic serum (ALS-IgG). ALS-IgG also suppressed the formation of circulating antibodies against rat thyroglobulin.     

The dual necessity for delayed hypersensitivity and circulating antibody in the pathogenesis of experimental allergic orchitis in guinea-pigs

1. Guinea-pigs injected with testis autoclavate (WT) in incomplete adjuvant develop antibody but no delayed hypersensitivity or testicular lesions. These animals fail to develop delayed hypersensitivity or a lesion when subsequently injected with antigen in complete adjuvant. Animals similarly treated, and then given repeated injection of immune cells from animals treated with whole testis and complete adjuvant, develop both delayed hypersensitivity and the characteristic orchitis.

2. Guinea-pigs injected with a purified fraction (designated H) with incomplete adjuvant fail to develop either circulating antibody, delayed hypersensitivity or a testicular lesion. Animals subsequently injected with this antigen and complete adjuvant, develop delayed hypersensitivity but no circulating antibody or testicular lesion. Three of the five animals similarly treated and given repeated injections of serum containing anti-testis antibody also develop orchitis.

3. A sequential study of fourteen animals killed at daily intervals after they were injected with WT and complete adjuvant, showed that antibody was first detected in the testis on the same day as delayed hypersensitivity first appeared. Circulating antibody was not detected until 2 days later. The specific testicular antigen showed first signs of disappearing on the 10th day, by which time histological evidence of orchitis was first detected.

4. Antibody was not detected in the testes in any animal in the absence of delayed hypersensitivity.

     

Cytophilic antibody in guinea-pigs with delayed-type hypersensitivity

Cytophilic antibody has been demonstrated in the serum of guinea-pigs injected with sheep red cells mixed with Freund's complete adjuvant. Sheep red cells adhered, in the absence of serum, to normal guinea-pig macrophages which had been treated in vitro with such serum. Treatment of polymorphs and lymphocytes with the antiserum did not confer on them this capacity to adsorb sheep red cells. The animals immunized with sheep red cells and complete adjuvant, when skin tested with an extract of sheep red cells, showed strong delayed-type reactions.

Cytophilic antibody was not detectable in the serum of the majority of guinea-pigs which received sheep red cells mixed with incomplete adjuvant. Skin tests in these animals produced strong Arthus reactions but no delayed reactions. Titres of haemagglutinating antibodies were similar whether the adjuvant was complete or incomplete.

     

Transfer of experimental autoimmune thyroiditis to normal syngeneic mice by injection of mouse thyroglobulin-sensitized T lymphocytes after activation with concanavalin A

Induction of autoimmune thyroiditis in normal syngeneic CBA/J mice was achieved by injection of 72-hr concanavalin A (Con A)-induced lymphoblasts from donor mice which had been immunized with mouse thyroglobulin (MTg) emulsified with complete Freund's adjuvant (CFA). Injection of lymph node or spleen cells, or frequent injection of serum taken from mice with autoimmune thyroiditis failed to transfer appreciable thyroiditis to recipient mice. Selection by treatment of incubated cells with monoclonal antibody and complement revealed that effector cells in Con A-induced lymphoblast populations for the transfer of autoimmune thyroiditis were Thy-1.2+, Lyt-1.1+, and Lyt-2.1- lymphocytes. These results demonstrate that experimental autoimmune thyroiditis can be adoptively transferred into naive mice by activated Thy-1+, Lyt-2- lymphoblasts.     

Migration inhibition of lymph node lymphocytes as an in vitro assay for cell-mediated immunity in the draining lymph nodes of parenterally immunized mice

This paper describes a method for in vitro measurement of specific cell-mediated immunity in the mouse. Animals were immunized parenterally with ovalbumin in Freund's incomplete or complete adjuvant, and a direct migration inhibition assay was performed, using lymphoid cells from the draining lymph nodes. Migration inhibition was found to be antigen specific, correlated with systemic delayed-type hypersensitivity measured in vivo by skin testing, and had a high degree of sensitivity for ovalbumin. The migrating cells were identified as lymphocytes. Lymph node lymphocyte migration inhibition provides a reliable in vitro assay for regional CMI in the mouse.     

Suppression of experimental allergic thyroiditis in guinea pigs by homologeous and heterologeous thyroglobulin

Summary Guinea pigs with experimental allergic thyroiditis, induced by three intradermal injections of thyroglobulin in complete Freund's adjuvant, were treated by daily injections of thyroglobulin emulsified in incomplete Freund's adjuvant over a period of 12 days. Besides lightmicroscopical, ultrastructural and immunohistochemical studies the serum antibody titers were determined and skin tests for delayed hypersensitivity were performed in all animals. The following results were obtained.1. Specific suppression of experimental thyroiditis by homologeous or heterologeous thyroglobulin resulted in almost complete absence of the inflammatory infiltrate in the thyroid glands.2. The cellular hyperactivity and the toxic-degenerative changes of the thyroid cells were still present in the desensitized animals. These changes were regarded as residues of the former inflammation of the glands.3. There was a good correlation between the presence of thyroiditis and delayed hypersensitivity in all animals. Such a correlation was not apparent between thyroiditis and antibody titers.The data, presented in this study, further support the role of cellular immunity in the pathogenesis of experimental allergic thyroiditis. The mechanism by which the suppressive effect is mediated is discussed.

---

Zusammenfassung Meerschweinchen mit einer experimentell induzierten Thyreoiditis wurde über einen Zeitraum von 12 Tagen täglich Thyreoglobulin, emulgiert in incomplettem Freundschem Adjuvans, injiziert. Neben licht- und elektronenmikroskopischen sowie immunhistochemischen Studien wurden Untersuchungen zur Überempfindlichkeit vom Spättyp an Hand von Hauttesten durchgeführt. Weiterhin wurde bei allen Tieren der Serum-Antikörpertiter gegen Thyreoglobulin bestimmt. Folgende Ergebnisse wurden erzielt.1. Eine spezifische Desensibilisierung durch homologes oder heterologes Thyreoglobulin führte bei den Versuchstieren mit experimenteller Thyreoiditis zu einem fast vollständigen Rückgang der Entzündung innerhalb der Schilddrüse.2. Das Fortbestehen stimulierter Follikelepithelien und toxisch-degenerativer Veränderungen in den Schilddrüsen desensibilisierter Tiere wurde als Hinweis für die vorausgegangene Entzündung gedeutet.3. Zwischen dem Vorhandensein einer Thyreoiditis und der Überempfindlickeit vom verzögerten Typ (Hauttest) besteht in allen Untersuchungsgruppen eine deutliche Korrelation. Eine solche Korrelation ließ sich nicht zwischen Thyreoiditis und Antikörpertiter nachweisen.Die Befunde stützen die Hypothese, daß die celluläre Immunität für das Zustandekommen der experimentellen Thyreoiditis den wesentlicheren Faktor darstellt. Die Mechanismen, die zur Desensibilisierung führen, werden in der vorliegenden Arbeit diskutiert.

---

---

This work was supported by the Deutsche Forschungsgemeinschaft.     

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.     

The effect of thyroid antigens on the in vitro migration of leucocytes from patients with Hashimoto thyroiditis

A total of fifty-two patients with Hashimoto thyroiditis were tested for delayed hypersensitivity to thyroid antigens using the leucocyte migration test. The percentage of patients showing abnormal migration in the presence of crude thyroid extract, thyroglobulin, thyroid mitochondria and thyroid microsomes was 75, 44, 54 and 34% respectively. Fifty-three control patients were studied concurrently with the same antigens and the percentage showing abnormal migration was 4, 6, 6 and 6% respectively. The antigenic activity of the mitochondrial fraction was not organ specific; both liver and kidney mitochondria interfered with the migration of leucocytes from patients with Hashimoto thyroiditis.     

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.     

Histological and serological features of experimental autoimmune thymitis in guinea-pigs

Guinea-pigs injected with thymus or muscle in complete Freund's adjuvant developed `experimental autoimmune thymitis'. Thymitis was defined by accumulations of lymphocytes around Hassall's corpuscles in the thymic medulla and by distortion and compression of reticulin fibres towards the cortico-medullary junction. Thymitis occurred in five of eight animals injected with thymus and four of four animals injected with muscle.

Immunized guinea-pigs showed delayed hypersensitivity responses to tissues used for immunization; these responses were not tissue specific. Circulating antibody responses, detected by immunofluorescence, did show specificity in that muscle induced the formation of antibody to the myoid antigen of skeletal muscle striations and to thymic myoid cells, and thymus induced antibody to the cytoplasm of lymphocytes and epithelial-reticular cells of the thymus.

Thymectomy 1 day before immunization did not influence the immune responses of immunized guinea-pigs, although we previously showed that it did prevent the development of a `myasthenic' neuromuscular block which occurs in animals with experimental autoimmune thymitis. It would appear that this block is not due directly to the delayed hypersensitivity or circulating antibody responses we have demonstrated, but to a humoral substance released from a thymus damaged by autoimmune thymitis.