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The effect of thymectomy on the course of experimental immune thyroiditis in mice Hay, John Bruce 1967

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THE EFFECT OF THYMECTOMY ON THE COURSE OF EXPERIMENTAL IMMUNE THYROIDITIS IN MICE JOHN BRUCE HAY B. Sc., The University of B r i t i s h Columbia,1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of ZOOLOGY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August,1967 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t he r e q u i r e m e n t s f o r an advanced d e g r e e a t t he U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Depar tment o r by h.i>s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l no t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f Z-<j-t>CO b-^j  The U n i v e r s i t y o f B r i t i s h C o l u m b i a Vancouve r 8, Canada Date ABSTRACT The purpose of t h i s project was to experimentally induce t h y r o i d i t i s i n the mouse and to examine any influence thymectomy might have on i t s development. Immunization was performed using homologous thyroid extract emulsified i n Freund's adjuvant. The mice were thymectomized within 18 hours of b i r t h using a suction technique. Results indicated that the incidence of thyroid lesions were reduced by 25 per cent with thymectomy. Serum antibody levels of an anti-thyroid nature were not reduced, however, as detected by a tanned c e l l haemagglutination technique. These results are i n accordance with recent evidence that the thymus i s important i n the development of c e l l u l a r immunity while immunoglobulin production i s dependent on a separate immune system probably involving the bursa of Fabricius i n chickens or i t s homologue i n mammals. i i . TABLE OF CONTENTS PAGE INTRODUCTION 1 MATERIALS AND METHODS 3 A. Animals 3 B. Thymectomy 3 C. Induction of Thyroiditis 5 D. Haemagglutinations 7 RESULTS 9 A. Histologic T h y r o i d i t i s 9-i ) Scoring of Thyroiditis 9 Comparison of Immunized Control, 1 0 Thymectomized and Sham-Operated Animals B. Thyroid Auto-Antibodies 1 ^ DISCUSSION 1 8 PLATE I (. Figures 1-k) 1 1 PLATE I I C Figures 5 - 8 ) 1 2 .PLATE I I I C Figure 9 ) 1 3 TABLE I 1 6 TABLE I I 1 7 LITERATURE CITED 2 8 i i i ACKNOWLEDGEMENTS I wish to express my gratitude to Dr. A.B. Acton for his introduction to the subject f i e l d and for his u n t i r i n g consideration. Also, I appreciate the assistance of Dr. C.V. Finnegan, and Dr. A. Perks for t h e i r c r i t i c i s m of the thesis. 1 INTRODUCTION Removal of the thymus i n newborn rodents leads to a depression of immunologic responsiveness ( M i l l e r , 1 9 6 l ; Good et a l , 1962 ). There i s a measurable decrease i n the c i r c u l a t i n g lymphocyte count as well as a reduced a b i l i t y to reject foreign' skin grafts. Serum antibody production i s also reduced with respect to some of the antigens that have been studied ( Fahey, et a l , 1 9 6 5 ; Humphrey, Parrot and East, 196^ ) . In the embryonic state the thymus i s the f i r s t organ to show a lymphoid appearance characterized by a large population of lymphocytes. Whether or not these thymus lymphocytes supply a l l of the other lymphoid tissues i s not yet determined, but ce r t a i n l y there i s a migration of lymphocytes from the thymus ( F i c h t e l i u s and Bryant, 1962 ). Considerable evidence also supports the humoral influence of thymus tissue on the development of immunologically competent c e l l s ( Osoba and M i l l e r , 196^; Levey, Trainin and Law, 1 9 6 3 ; Aisenberg and Wilkes, 1965 ) . The production of inflammatory lesions, consisting primarily of lymphocytes, by i n j e c t i o n of the appropriate organ extract emulsified i n a mixture of o i l and b a c t e r i a l proteins ( Freund's adjuvant ) i s a well established phenomenon ( See Whipple, I 9 6 5 )• One such condition i s experimental t h y r o i d i t i s , the development of which i s considered to be a consequence of auto-immunity. Antibodies are synthesized to react against " s e l f " antigens and lesions containing large numbers of lymphocytes are observed i n the thyroid ( Terplan, Witebsky et a l , i 9 6 0 ) . I t would seem possible, therefore, that the thymus may exert some influence on the production of these auto-antibodies and lymphocytic lesions. The purpose of t h i s investigation was to study the effects of thymectomy i n newborn mice on the development of experimental immune t h y r o i d i t i s . Recently, similar investigations have been carried out on guinea-pigs ( Lepulescu, 1 9 6 5 ) and chickens ( Jankovic, I 9 6 5 ) • 3 {MATERIALS AND METHODS A. Animals The mice used were randomly bred from a Swiss albino colony maintained at the University of B r i t i s h Columbia for f i f t e e n years. L i t t e r s were weaned at three weeks and fed mouse p e l l e t s and water Sid libitom . The study included 17 thymectomized, 8 sham-operated and 17 control animals. Both sexes were selected at random. B. Thymectomy Thymectomy and sham operations were performed within eighteen hours of b i r t h . The method followed was that c3£ Osoba ( 1966 ). With experience the operative mortality was reduced to approximately ten per cent but the survival at the end of seven weeks approached f i f t y per cent. Thymectomized animals are more susceptible to i n f e c t i o n and some of the f a t a l i t i e s may be associated with a wasting condition or runt disease ( Parrot and East, 1962 ) although t h i s doesn't usually occur u n t i l after seven weeks of age. I n i t i a l l y , maternal neglect and cannibalism severely reduced the survival rate. Several methods were attempted to a l l e v i a t e t h i s , including the u t i l i z a t i o n of foster mothers; the addition of Librium ( Roche ) tr a n q u i l i z e r to the drinking water; the use of mentholated vaporub ( Vick's) on the i n c i s i o n and around the cage to interfere with the olfactory s e n s i t i v i t y of the mother and the u t i l i z a t i o n of a p l a s t i c spray bandage ( Aeroplast ). The highest survival rate, however, was obtained after i n j e c t i n g the mother with 0.15 cc. of a 2:1 d i l u t i o n of saline and Nembutal. The anaesthetized mother was then.left i n the cage and the operated l i t t e r returned before the effect of the anaesthetic disappeared ( approximately 1 hour ). Also, i t was much more effective to use experienced mothers. i . e . those having borne one or two previous l i t t e r s . Newborn animals were exposed to a f o i l covered ice block i n an insulated chamber u n t i l body movements had ceased ( U—5 min. ). The animalfe extremeties were secured with e l a s t i c on an animal board. Reasonable s t e r i l e technique was observed by keeping instruments i n alcohol and the operative area swabbed. A straight i n c i s i o n through the skin was made from the area of the si x t h r i b to the neck region anterior to the salivary t i s s u e . Using a cotton swab the salivary tissue was then retracted anteriorly. I r i s scissors were inserted under the c l a v i c l e and p a r a l l e l incisions were made on either side of the sternum taking care to avoid major blood vessels since loss of a single drop of blood was usually f a t a l . The sternal cartilage was then completely removed exposing both lobes of the thymus. The t h i n f a s c i a surrounding the gland was then removed using jewellers forceps. Gentle, controlled suction was applied with a mechanical vacuum pump f i t t e d with a c a p i l l a r y t i p . After unbinding the forelimbs to 5 close the chest cavity, two single.stitches were made through the skin with 7-0 s i l k , 3/8 c i r c l e sutures. The animals were incubated under a lamp then returned to the mother. Sham-operated animals were subjected to the same procedure. The sternum was removed but the thymus was l e f t i n t a c t . The animals s a c r i f i c e d at seven weeks were examined both macroscopically and microscopically ( medio-sternal tissue remnants were f i x e d , stained and sectioned ) for residual thymus. These animals are described i n the r e s u l t s . Since the thymectomy technique involved surgical manipulation near the area of the thyroid the p o s s i b i l i t y of tissue injury was checked by h i s t l o g i c examination of a normal, thymectomized thyroid at seven weeks. C. 'Induction of •'Thyroiditis Mouse thyroids were collected and frozen. A crude extract was prepared by homogenizing the thyroid tissue i n a Potter-type tissue homogenizer and a equal volume of isotonic mammalian saline was added. The extract was then.centrifuged to separate the s t i l l s o l i d tissue from the supernatant which was used as the antigen. Approximately one hundred mice were prepare 2 ml. of• such an extract. Thyroiditis was f i r s t produced using rats C R o i t t , l'96l ) to test the technique since the i n i t i a l attempts with mice were 6 unsuccessful. 1 A t r i a l and error method of inducing h i s t o l o g i c thyroid lesions was used for the mice since the procedure was not described elsewhere. This involved injections at intervals varying from one day to two weeks apart and sacrificed, at similar i n t e r v a l s . Various concentrations of antigen were attempted. For the mice used, the most successful results were obtained by using a 0.05 cc. in j e c t i o n of thyroid antigen emulsified ( by rapidly drawing and expelling through a.B.D. 2k needle ) i n an equal volume of complete Freund's adjuvant ( Difco ). Injections were made near the base of the t a i l taking care to avoid major blood vessels. This allowed for a slow d i f f u s i o n of antigen over a period of a few days. A similar booster was given at one week and s a c r i f i c e was made two weeks after the i n i t i a l i n j e c t i o n . Autopsy was performed on Nebutal anaesthetized animals and the trachea with surrounding pretracheal muscle associated thyroid tissue were fix e d i n formal saline. The precaval veins were severed and the blood was collected using heparinized pipettes. Serum was stored at -10° C for haemagglutinations. The thyroid tissue was dehydrated i n alcohol, cleared i n benzene and embedded i n pa r a f f i n . S e r i a l sectioning was done at 5 u and the material was stained with Ehrlich's haematoxylin and counterstained with alcoholic eosin. D. Haemagglutinations 7 D. Haemagglutinations Tanned c e l l haemagglutinations were performed on the sera according to the method described by Fulthorpe, R o i t t , et a l ( 1 9 6 l ). Sheep red blood c e l l s were washed and suspended i n a phosphate buffer pH 7-2 to a f i n a l concentration of 1.0%. These c e l l s were treated with tannic acid diluted 1:20,000. One half of them were coated with antigen while the remaining half was used for control t e s t s . Since mouse thyroglobulin i s not available commercially, a p a r t i a l l y p u r i f i e d extract was prepared by ammonium sulphate fractionation similar to the method of K i t e , Argue, and Rose ( 1 9 6 6 ) for monkey thyroids. From a pool of mouse thyroids a crude extract was prepared i n the manner described above. This was centrifuged at 68,000 g i n a Spinco Model L ultracentrifuge with a #50 rotor for 45 minutes to produce a c l a r i f i e d extract. Enough neutralized lt.0 M ammonium sulphate solution was added drop by drop at room temperature with constant s t i r r i n g to bring the f i n a l concentration to 2.k M. The small precipitate that formed was removed by centrifugation. The concentration was then adjusted to 2.0 M and the white precipitate that resulted was washed twice with 2.0M ammonium sulphate and then dissolved i n 2 ml. of phosphate buffered saline. Overnight d i a l y s i s against the same phosphate buffered saline resulted i n 3 m l . of thyroglobulin solution. Protein determination by the Lowry method showed 2 .8U mg./ml. This was brought to a 0.1% protein concentration i n phosphate buffer and was 8 used—as—the--ant-i-gen—to-coat the tanned sheep red c e l l s . By-adding 0.2% formalin to the c e l l s , they are usable for up to nine months. Serum dil u t i o n s of 1:5 to 1:25 m i l l i o n were set up and 0.1 ml. of c e l l s were added to 0.1 ml. of each serum d i l u t i o n . Controls for each serum sample were done using uncoated sheep red c e l l s . Haemagglutinations were attempted i n depression slides but the shape of the concavity made readings impossible so haemagglutination tubes were used. 9 RESULTS The nature of the investigation permitted a semi-quantitative analysis only. The two c r i t e r i a selected to represent the experimental autoimmune condition were the incidence and severity of thyroid lesions and the detection of serum auto-antibodies. They are represented separately below. A. Histologic Thyroiditis ( 1 ) Scoring of Thyro i d i t i s Arbitrary c r i t e r i a for id e n t i f y i n g the severity of the thyroid lesions were established as described below. Figure 1 represents the appearance of normal mouse thyroid ti s s u e , i . Grade I-mild t h y r o i d i t i s C h a r a c t e r i s t i c a l l y there was a reduction i n f o l l i c l e size with leucocytes adhering to c a p i l l a r y walls and some perivenular lymphocyte accumulation. ( See Figure 2 ) This condition i s similar to that described for lesions produced by Freund's adjuvant alone ( Pearson, Waksman and Sharp, 1 9 6 l ) . i i . Grade I I - d e f i n i t e t h y r o i d i t i s In t h i s instance there i s substantial c o l l o i d l o s s . E p i t h e l i a l f o l l i c l e c e l l s are often sloughed off ( Figure 3 ) and are seen i n the lumen together with large numbers of foreign c e l l s . Mononuclear c e l l s are the most prevalent and macrophages can be i d e n t i f i e d . Polymorphonuclear leucocytes are present and often the 10 - f o l l i c l e — i s f i l l e d with a great deal of c e l l u l a r debris. (See Figure k ) In t h i s condition up to one-half of the gland ( either or both lobes ) was occupied with inflammatory f o c i , i i i . Grade Ill-severe t h y r o i d i t i s The normal architecture i s almost completely lost' and the f o l l i c l e s are widely separated by fibrous lymphocytic stroma. Intact f o l l i c l e s contain l i t t l e c o l l o i d and invaded f o l l i c l e s often remain as c e l l u l a r islands, ( Figure 5 ). More than one-half of the gland was i n f i l t r a t e d and the invasive c e l l s occupied the surrounding connective and muscle t i s s u e , ( Figure 6 , 7 and 8 ) . ( 2 ) Comparison of Immunized Control, Thymectomized and Sham-Operated Animals A comparison of the induced thyroid lesions i s represented i n Table 1. The control animals, Group I , were l i t t e r s selected at random and immunized on the f i f t h and seventh week. Fourteen of the seventeen animals ( 8 2 %) showed recognizable thyroid changes as described. The mean severity of the lesions was 2 . 2 . Thymectomized animals were divided into three classes depending on the residual thymus tissue recognizable upon autopsy. Considering the twenty-one animals i n Group I I that underwent neonatal thymectomy, immunization and subsequently survived to seven weeks of age, f i f t e e n ( Group I I I ) showed only a microscopic remnant or complete thymectomy. Group IV comprised seven animals that had no trace of residual thymus. The percentage of animals exhibiting t h y r o i d i t i s was reduced by f i f t e e n to twenty-five per cent 11 PLATE I Figure 1. Normal mouse thyroid f o l l i c l e s XI,000. Figure 2. Grade I t h y r o i d i t i s with perivenular leucocyte accumulation and m i c r o f o l l i c u l a r architecture. X200. Figure 3. Definite t h y r o i d i t i s . E p i t h e l i a l ' c e l l s of the f o l l i c l e are being sloughed o f f into the lumen. Xl800. Figure k. C e l l u l a r i n f i l t r a t e of a f o l l i c l e showing mononuclear c e l l s , macrophages, and c e l l u l a r debris. X1700. 12 PLATE I I Figure 5- Definite t h y r o i d i t i s . ' Normal tissue structure i s l o s t and remaining f o l l i c l e s appear as c e l l u l a r • islands of i n f i l t r a t e d c e l l s . X200. i. Figure 6 . Definite t h y r o i d i t i s with f o l l i c l e s i n various stages of i n f i l t r a t i o n by lymphocytes. X200. Figure 7* Mononuclear c e l l s have invaded muscle and connective tissue surrounding the thyroid. Grade I I I t h y r o i d i t i s . Xl ' 6 0 . Figure 8. Grade I I I t h y r o i d i t i s . Low power of one thyroid lobe showing mononuclear c e l l s i n surrounding muscle and connective tissue. XhO. 13 PLATE I I I Figure 9« Distorted shape of a lymphocyte penetrating the endothelium of a venule i n perithyroid muscle. X2,000. Ik in_tJie_t.hvme£:tomized animals. Moreover, the mean severity of the t h y r o i d i t i s was reduced to 1 . 7 and 1 . 3 i n the thymectomized groups. Group V represents the eight sham-operated animals a l l exhibiting severe t h y r o i d i t i s . Histologic examination of the thyroid of a non-immunized, thymectomized animal revealed no trace of abnormal thyroid tissue which may have resulted from thymectomy per se,or from damage during the operative procedures. B. Thyroid Auto-Antibodies The results of the tanned c e l l haemagglutinations for thyroid auto-antibodies are summarized i n Table 2 . T i t r e values were taken as the highest value that showed positive agglutination. Using control, uncoated red blood c e l l s a l l the sera showed a negative response. Serum was not collected from a l l the animals referred to i n the hi s t o l o g i c study. Some of the t i t r e s were greater than 1 : 2 5 x 1 0 ^ and were not taken to the endpoint. This test i s primarily of diagnostic value for auto-antibodies and i s used extensively i n studies of th e o r e t i c a l problems associated with auto-immunity. In a study involving mice i t was particular\y valuable since only a few drops of serum are required. Moreover, the test i s 1 , 0 0 0 times more sensitive than the pr e c i p i t a t i o n reaction. Direct agglutination t i t r e s do not correlate perfectly with antibody levels as determined by quantitative techniques. Fulthorpe, R o i t t , Doniach and Couchman ( 1 9 6 l ) have compared anti-thyroid antibody 15 levels determined by radioactive coprecipitation ( Roitt and Doniach, 1959 ) and quantitative p r e c i p i t a t i o n methods ( Heidel-berger and Kendall, 1935 ) with direct haemagglutinations. High haemagglutination t i t r e s do correlate with high serum antibody concentrations. There i s , therefore, no s i g n i f i c a n t difference between the anti-thyroid antibody t i t r e s i n the control and thymectomized groups. A l l animals showed a high t i t r e diagnostic of auto-antibodies. Four animals that showed no trace of hi s t o l o g i c t h y r o i d i t i s had anti-thyroid antibody t i t r e s higher than 1 : 5 0 0 , 0 0 0 . Also, f i v e animals with no residual thymus tissue had auto-antibody t i t r e s of 1 : 2 5 0 , 0 0 0 or higher. HISTOLOGIC THYROIDITIS COMPARING CONTROL, SHAM-OPERATED AND THYMECTOMIZED ANIMALS TREATMENT NUMBER OF ANIMALS SEVERITY OF LESIONS +• + + + + + MEAN SEVERITY PER CENT THYROIDITIS GROUP 1 IMMUNIZED CONTROL 17 3 1 2 11 2.2 82 GROUP II T O T A L T H Y M E C T O M I Z E D 21 7 2 3 9 1.7 67 GROUP l l l A T H Y M E C T O M I Z E D . (<10°/ o RESIDUAL THYMUS ) 15 6 2 1 6 1.7 60 GROUP IV T H Y M E C T O M I Z E D (NO RESIDUAL T H Y M U S ) 7 3 1 1 2 1.3 57 GROUP V S H A M - OPERATED 8 0 0 0 8 3.0 100 ^ INCLUDES AN IMALS WITH N O RESIDUAL THYMUS AS REPRESENTED IN G R O U P IV TABLE I HAEMAGGLUTINATIONS SERUM DILUTION 25-2 50~2 25 " 3 50 " 3 25 " 4 50 - 4 25" 5 50" 5 25 " 6 U N C O A T E D C O N T R O L CELLS IMMUNIZED CONTROL 1 2 1 2 A 5A — TOTAL T H Y M E C T O M I Z E D 1 3 1 4 A 3 A — S H A M - OPERATED 2 — A HIGHEST TITRE RECORDED H TABLE II DISCUSSION 18 The importance of the thymus i n the development of the immune system has been the subject of many investigations and recently more than one symposium, ( See: The Thymus i n Immuno-biology, 1 9 6 U , Good and Gabrielson,-editors; The Thymus, 19'6U -Defendi, editor; and The Thymus: Experimental and C l i n i c a l Studies, 1966 - Wolstenholme and Porter, editors. ) t The thymus appears to be the centre of lymphoid-cell p r o l i f e r a t i o n and d i f f e r e n t i a t i o n ( M i l l e r , 196I+ ). The exact nature of the influence of the thymus i s not e n t i r e l y clear, but there i s substantial evidence for a humoral factor or factors. Such studies have been done on mice, ( Osoba and M i l l e r , I 9 6 U and Levey, Trainin and Law, 1963 ) on rabbits ( Trench, Watson et a l , I 9 6 6 ) and on hamsters ( Wong et a l , 1965 ) • I t i s also established that the immune system i s not always a normal, b e n e f i c i a l homeostatic mechanism. A state where there i s an abnormal immunological reaction against self-antigens may be defined as auto-immunity ( Dameshek, 1966 ). I t would seem possible then, that the functioning of the thymus may influence autoimmunity. Autoimmunity may be associated with antigens that are normally i n isolated regions which become accessible to the immune system only after i n j u r y , possibly after the immune system has d i f f e r e n t i a t e d , and are therfore recognized as foreign. Such may 19 be the_case with thyroglobulin ( Rose et a l , 1 9 6 5 ), seminiferous spermatozoa ( Feltkamp et a l , I 9 6 5 ), lens of the eye ( Halbert, Manski and E h r l i c h , 1 9 6 5 ) and elements of the central nervous system faaksman, I965 )• The second broad p o s s i b i l i t y for autoimmune development may involve an aberrant recognition system by abnormal immunocytes. Dameshek, ( 1 9 6 3 ) uses t h i s term to describe a complex of c e l l s p r o l i f e r a t i n g i n response to an antigen. There i s considerable evidence to show that a normal immune response follows the following sequence of events. The macrophages phagocytose foreign material which contain antigen and digest i t ( Roberts and Haurowitz, 1 9 6 2 ) . Material or information derived from t h i s may be picked up by small lymphocytes which have been shown to gather about the macrophages ( Sharp and Burwell, i 9 6 0 ) and i n response to the antigen these lymphocytes are transformed into large, primitive looking blast c e l l s or immunoblasts ( Tanaka et a l , I 9 6 3 ) which possess large numbers of ribosomes. The subsequent p r o l i f e r a t i o n of the blast c e l l s i s then considered to lead to the formation of plasma c e l l s ( involved i n serum antibody production ) and lymphocytes, (involved i n cell-mediated immunity ) or both, although experimental evidence i s s t i l l uncertain. Following the immune reaction, memory c e l l s are thought to remain , capable of rapidly reacting to the same antigen i f i t i s encountered again. According to Gowans and Mc Gregor, 0-965 ) these are almost surely the lymphocytes. 20 Autoimmunity as a consequence of some abnormality i n t h i s immunocyte complex has been hypothesized by Burnet ( 1959 ) i n his " forbidden clone " concept. He suggests that a somatic mutation could lead to the p r o l i f e r a t i o n of a clone of c e l l s that lose t h e i r immunologic tolerance to self-antigens. Such a si t u a t i o n could be i n i t i a t e d i n the thymus. This speculation, resulted from the appearance of germinal centres within the thymus associated with certain known autoimmune disorders. In contrast to other lymphoid organs the thymus usually lacks germinal f o l l i c l e s . However, haemolytic anaemia which develops spontaneously i n NZB/BL mice ( Bielchowsky, Helyer and Howie, 1959 ) and myasthenia gravis ( Castleman and. Norris, 19^-9 ) have been associated with thymic germ-i n a l centres. This p a r t i c u l a r anaemia can be transmitted from mouse to mouse by in j e c t i n g spleen.cells into the animals ( Holmes, Gorrie and Burnet, 1 9 6 l ) which might be the'forbidden clones" of Burnet's hypothesis. More recently, the effects of thymectomy have been studied on various animals with autoimmune disorders. In adult mice even, thymectomy near the time of immunization reduced the severity of a l l e r g i c aspermatogenesis ( Vojtiskova and Pokorna, I 9 6 U ) . In auto-immune strains of mice ( Howie and Helyer, 1966 ) neonatal thymectomy causes the precocious development and an increase i n acuity of the disorder. Moreover, normal thymectomized mice grafted with autoimmune thymus develop serological and h i s t o l o g i c a l evidence of auto-immune disease. 21 In b i r d s , the bursa of Fabricius seems to be a primary lymphoid organ that shares some of the attributes of the mammalian thymus ( Glick et a l , 1 9 5 6 ; Mueller, et a l , I 9 6 2 ). Neonatal thymectomy i n chickens as wel l as i n mammals considerably retards the rejection of skin homografts ( Warner, I 9 6 2 ) despite.seemingly normal antibody production. However,.surgical or hormonal bursectomy:suppresses the antibody forming capacity of chickens. Bursal homografts i n such animals restores the impaired antibody response ( Jankovic et a l , 1963 ) . Jankovic and workers ( I 9 6 3 ) have also shown that neonatal thymectomy i n chickens does s i g n i f i c a n t l y reduce experimental a l l e r g i c encephalomyelitis. This disease can be produced equally well i n bursectomized and normal chickens by i n j e c t i o n of spinal cord i n complete Freund's adjuvant. Another experimental.autoimmune condition studied i n chickens i s induced t h y r o i d i t i s ( Jankovic et a l , 1965 ) produced by i n j e c t i o n of homologous thyroid tissue i n complete Freund's adjuvant. Again, the.severity of the lesions was reduced by thymec-tomy performed at hatching while bursectomy had no eff e c t . Auto a n t i -body determination however, was not studied. This.evidence strongly suggests that the aggressive agent i n the development of predominantly mononuclear c e l l lesions associated with both induced a l l e r g i c encephalomyelitis as wel l as t h y r o i d i t i s i s the lymphocyte and that the autoantibody i n the.serum i s of less obvious significance. 22 I-n—the'present study the results demonstrate a.definite reduction i n the severity of t h y r o i d i t i s lesions following neonatal ' thymectomy i n mice. This i s most probably a direct consequence of the lymphocytopenia r e s u l t i n g from thymectomy. This type of mononuclear c e l l invasion occurring i n conjunction with a reaction of the delayed hypersensite type ( R o i t t , I 9 6 7 ). Dermal reactions to homologous mouse thyroid extract were attempted, but a.quantitative determination of the reaction both macroscopically and h i s t o l o g i c a l l y was rather unsatisfactory for any comparison to be made. Spiegelberg and Miescher, ( 1963 ) used the immunosuppressive drug, 6-mercaptopurine. They were able to.reduce the delayed response to thyroglobulin and also reduce the severity of t h y r o i d i t i s without s i g n i f i c a n t l y affecting the antibody t i t r e . In Jankovic's studies ( I 9 6 5 I with t h y r o i d i t i s and a l l e r g i c encephalomyelitis, the delayed hypersensitivity response to thyroglobulin and spinal cord antigens was also reduced with thymectomy. Roitt and Doniach ( I 9 6 7 ) claim that delayed hypersensitivity reactions probably operate i n the pathogenesis of many of the experimentally induced auto-immune.conditions i n animals. However, the.evidence i s not nearly so convincing for the human auto-immune diseases. A synergism.between cell-bound antibodies associated with the lymphocytes and serum antibodies i s a p o s s i b i l i t y that has yet to be determined. The early changes. i n a delayed hypersensitivity ..reaction following intradermal i n j e c t i o n of an antigen consist of perivenular cuff ing of lymphocytes derived from the blood.Gowans, ( 1962 ) shows 2 3 that emigration of—lymphocytes from blood'into lymph nodes takes place from the venules and i n pathological conditions the migration i s again from the venules. This i s i n contrast to the e s s e n t i a l l y polymorphonuclear leucocyte.response seen i n the Arthus reaction, which depends upon the p r e c i p i t a t i o n of antigen-antibody complexes within the vessel w a l l . Arthus or immediate reactions occur soon after i n j e c t i o n of antigen. They can be passively transferred by serum. Delayed reactions, however, reach a maximum at about 2h hours. The inflsaed area i s f i l l e d with mononuclear c e l l s and the condition cannot be transferred v i a the.serum. The photomicro-graphs described on page 1 1 also demonstrate a similar perivenular lymphocyte penetration. The c e l l shape of the lymphocyte i s extremely p l i a b l e as the micrograph ( Figure 9 ) indicates. This i s also evident i n cinematographic studies of emperipolesis... i n lymphocyte-thyroid culture ( Acton,I96U ) . Jankovic ( I 9 6 5 ) describes, with photomicrographs, direct p a r t i c i p a t i o n of thymus lymphocytes i n the invasion of the thyroid. Approximately 65% of normal chickens show small thymic lobes either i n the adipose tissue of the thyroid area or d i r e c t l y attached to the thyroid gland, separated only by a t h i n layer of connective tissue. In cases of t h y r o i d i t i s the thymus-thyroid bar r i e r has disappeared and apparent massive migration of mononuclear c e l l s d i r e c t l y from the thymus cortex to the thyroid can be seen. There was however, no evidence of.germinal centres or inflammation within the thymus i t s e l f . 2h The-mul-t-iple antigenicity of the thyroid extract prepared for immunization may have affected the areas of tissue that showed c e l l u l a r i n f i l t r a t e s . Such a crude extract contains a very large number of antigenically d i s t i n c t proteins. Thus, parathyroid proteins may he present as well as various muscle and connective tissue proteins. This could account for the mononuclear c e l l lesions i n the muscle tissue described as Grade I I I t h y r o i d i t i s . Jankovic ( 1965 ) shows the concommital occurence of para t h y r o i d i t i s with the development of thyroiditis;> Tolnai ( 1966 ) was able to demonstrate• mononuclear c e l l invasion near the i n j e c t i o n s i t e at l e a s t , of homologous ske l e t a l muscle extract emulsified i n Freund's adjuvant. It should be emphasized however, that the antigen used for the haemagglutinations was a more highly p u r i f i e d thyroglobulin product and that cross-reactions with antibodies other than thyroglobulin auto-antibodies should therefore have been reduced. Further information regarding the aggressive p a r t i c i p a t i o n of the lymphocyte has been accumulated from passive transfer experiments. A l l e r g i c encephalomyelitis has been induced passively by the inocula-t i o n of lymph node c e l l s taken from animals immunized with brain tissue i n Freund's adjuvant under conditions where the transferred lymphoid c e l l s were able to survive ( Astr'om and Waksman, 1963 ) • P a r a l l e l experiments using large volumes of serum from immunized animals have f a i l e d to produce lesions. As yet, passive transfer of lymphoid c e l l s has not successfully induced experimental t h y r o i d i i s . 25 The results outlined i n t h i s text indicate that a high t i t r e of thyroid auto-antibody does not necessarily imply the co-existence of lesions i n the gland i t s e l f . Rose et a l ( 1965 ) using rabbits injected with alum-precipitated thyroid extract demonstrated that they possessed high t i t r e s of c i r c u l a t i n g antibody for long periods of time, but develop no thyroid lesions nor s i g n i f i c a n t delayed hypersensitivity. Mc Master, Lerner, and Exum ( I 9 6 I ) attempted to correlate:;the severity of lesions with the delayed hypersensitivity reaction and antibody l e v e l s . For example, i n guinea pigs immunized with thyroglobulin and incomplete Freund's adjuvant low levels of thyroglobulin antibody could be detected without inflammatory changes i n the gland or i n dermal reactions. Again, i t should be noted that one or more features of autoimmunity may be acting s y n e r g i s t i c a l l y or they may be separated i n time only. A detailed history of the disease i n mice was not madeafter f i v e weeks of development. The use of Freund's adjuvant i s common i n the experimental production of autoimmunity. By emulsifying small concentrations of the antigen i n complete Freund's adjuvant which contains k i l l e d Mycobacterium severe pathological manifestations and c i r c u l a t i n g auto-antibodies are consistently produced. However, i n a few cases, lesions have been induced without the p a r t i c i p a t i o n of adjuvant. Levine and Wenk, ( 1965 ) have shown that a number of strains of rats w i l l succumb to experimental encephalomyelitis when injected with homologous nervous tissue only, but the dose required was 200 - kOO times larger than with adjuvant. Thyroiditis has been induced i n r 26 rabbits by using heterologous thyroglobulin ( Terplan, Witebsky, Rose, Paine and Egan, i 9 6 0 ). The mechanism by which the adjuvant enhances the immunologic reaction i s not c l e a r , but possibly the mycobacterium somehow acts to i n i t i a t e the d i f f e r e n t i a t i o n of the immunocyte complex allowing for a more widespread reaction to the homologous antigen. The findings represented i n t h i s study are i n . accordance with a similar study performed on young guinea-pigs by Lupulescu et a l ( 1965 ) insomuch as the reduced nature of h i s t o l o g i c thyroid lesions with thymectomy i s concerned. They, however, were unable to detect any c i r c u l a t i n g antibodies by the complement f i x a t i o n method i n thymectomized animals. Also i n the immunized animals c i r c u l a t i n g antithyroid antibodies were not detected regularly and the t i t r e varied. This i s i n sharp.contrast to the present finding and i t may be a consequence of the detection methods used. Rose et a l ( I 9 6 5 ) were able to detect antibodies to thyroglobulin using the tanned c e l l haemagglutination method i n monkeys, but were able to produce l i t t l e or no complement f i x a t i o n with soluble thyroid proteins. The phylogeny of the immune system has been extensively reviewed by Papermaster et a l ( 1962 )• The dissociation of c e l l u l a r immunity which i s dependent on the thymus from immunoglobulin development on the bursa i n birds has also been established. This has promoted attempts to describe a mammalian equivalent to the bursa. According to Good and workers ( 1966 ) the " Primordial tissue for the mammalian immuno globulin producing system i s presumed to be another gut-associated 27 glandular-lympho-id t i s s u e , lympho-epithelial i n type,'.relatively large i n the embryo and neonate, and involuting at about the time of sexual maturity. " Candidates so far include pharyngeal and palatine t o n s i l s and recently Cooper, Paterson and Good ( 1966 ) obtained evidence that the rabbit•appendix and Peyer's patches.function l i k e a bursa. With these considerations plus preliminary r r e s u l t s on DNA synthesis rates i n the gut epithelium of ,guinea-pigs which show exceedingly high rates, comparable to the thymus and bursa, F i c h t e l i u s ( I967 ), postulates that the entire gut-epithelium of mammals may function i n immunoglobulin development as does the avian bursa of Fabricius. This investigation . seems to support the previously reported evidence ( F i c h t e l i u s , I967 ) suggesting that the immunoglobulin synthesizing system may be somewhat divorced from the system concerned with c e l l u l a r immunity. Thymectomy d e f i n i t e l y reduced the c e l l u l a r manifestations of induced t h y r o i d i t i s while the immunoglobulin le v e l s were seemingly unaffected. I t would appear, also, that the existence of thyroglobulin auto-antibodies i s not i n i t s e l f diagnostic of t h y r o i d i t i s . This does not exclude the p o s s i b i l i t y 7 that the auto-antibodies preview the development of thyroid lesions. Some of the unanswered aspects of autoimmunity further studied i n animals with completely dissociated' thymus and bursa dependent immune systems. Future studies on lower vertebrates may prove to be worthwhile. 28 LITERATURE CITED Aisenberg, A.C. and Wilkes,;B. 1 9 6 5 . P a r t i a l immunologic restoration of neonatally thymectomized rats with thymus containing d i f f u s i o n chambers. Nature,( Lond.) 2 0 5 _ : 7 l 6 . Astrom',K.E. and Waksman,B.H. 1962.- The passive transfer of experimental a l l e r g i c encephalomyelitis and n e u r i t i s with l i v i n g lymphoid c e l l s . J . Path. Bact., 83_: 8 9 . Bielschowsky, M., Helyer,B.J. and Howie,J.B. 1959- Spontaneous hemolytic anemia i n mice of the NZB/BL s t r a i n . Proc. Univ. Otago Med. Sch. 3 7 : 9 . Burnet,F.M. The clonal selection theory of acquired immunity. 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