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[39] Autoimmune orchitis and thyroiditis
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TABLE III GLOMERULAR ANTIBODY BINDING (GAB) AND URINE PROTEIN EXCRETION 24 fir AFTER ADMINISTRATION OF SHEEP ANTI-RABBIT GBM TO NORMAL AND C6-DEFICIENT (C6D) RABmTSa
Groupb
GABc (gg IgG/104glomeruli)
Urine protein excretion (mg/g creatinine)
Normal (5)d C6D (6)
15.8 + 2.71e 18.7 + 1.99
42.0 + 26.Y 5.1 + 5.5
From Groggelet al. 44 b Rabbits are intravenously injected with sheep anti-rabbit GBM IgG (12 mg/kg) labeled with 125I. c Since kidneys are not perfused with saline, GAB is corrected for nonspecific binding by the double-isotope technique using nonimmune sheep 13'I-IgGas a plasma marker. a Number of rabbits studied is given in parentheses. e Values are means + SD.
a
development o f heterologous-phase proteinuria. 44 Urine protein excretion is measured for 24 hr after antibody injection, and glomerular binding is assessed by the dual-isotope m e t h o d at the end o f the urine collection period. The results show a lower rate o f urine protein excretion in the C6-deficient group, which cannot be attributed to their having less glomerular antibody. Acknowledgments This work was supported by U.S. Public Health Service Grant RO1 DK30932 from the National Institute of Diabetes, Digestive and Kidney Diseases, by an American Heart Association Established Investigator Award (D.J,S.), and by the Kidney Foundation of Canada and
Medical Research Council of Canada (A.V.C.).
[39] Autoimmune Orchitis and Thyroiditis By PIERLUIGI
E. BIGAZZI
Introduction Studies o f organ-specific a u t o i m m u n e diseases occurring in laboratory animals have provided useful and often illuminating information regarding the etiologic and pathogenetic mechanisms o f h u m a n a u t o i m m u n e disease. A u t o i m m u n e disease can be induced in animals by the administraMETHODS IN ENZYMOLOGY, VOL. 162
Copyright© 1988by AcademicPress,Inc. All fightsof reproductionin any form~erved.
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tion of drugs and other chemicals 1 or X-irradiation and by surgical manipulations (e.g., thymectomy). More commonly, such experimental models of autoimmunity are obtained by the immunization of laboratory animals with homologous or heterologous organ-specific antigens, usually emulsified in Freund's complete adjuvant or in association with other adjuvants (e.g., Bordetella pertussis). 2,3 However, as Burnet once noted, "An elderly woman with Hashimoto's disease of the thyroid has not been injected with rabbit thyroid in Freund's complete adjuvant." 4 In other words, there is a need for animal models of autoimmune disease that are not dependent on the artificial administration of antigens and adjuvants and that resemble more closely the situation occurring in humans. For this reason, we have suggested that careful investigation of spontaneous organ-specific autoimmune disease of animals may provide additional insights into human iUness. 5 In this chapter, we review both an experimentally induced and a spontaneous model of organ-specific autoimmune disease. Postvasectomy Autoimmune Orchitis Autoimmune orchitis is induced experimentally in a variety of animal species by immunization with spermatozoa or testicular antigens, emulsified with Freund's complete adjuvant or associated with B. pertussis. 6 However, there is one model of autoimmune orchitis occurring in rabbits, initiated by occlusive surgery of the vas deferens (vasectomy) and not dependent on the administration of exogenous adjuvants. 7-1° In the following section we describe in detail the procedures employed in our laboraP. E. Bigazzi, in "Immunotoxicology and Immunopharmacology" (J. Dean, M. I. Luster, A. E. Munson, and H. Amos, ¢ds.), p. 277. Raven, New York, 1985. 2 p. E. Bigazzi and N. R. Rose, in "The Autoimmune Diseases" (N. R. Rose and I. R. Mackay, eds.), p. 161. Academic Press, Orlando, Florida, 1985. 3 p. E. Bigazzi, in "Autoimmunity and Endocrine Disease" (R. Volp¢, ed.), p 345. Dekker, New York, 1985. 4 F. M. Burnet, "Auto-immunity and Auto-immune Disease," p. 101. Davis, Philadelphia, Pennsylvania, 1972. s p. E. Bigazzi and N. R. Rose, Prog. Allergy 19, 245 (1975). 6 K. S. K. Tung and A. C. Menge, in "The Antoimmune Diseases" (N. R. Rose and I. R. Mackay, eds.), p 537. Academic Press, Orlando, Florida, 1985. 7 p. E. Bigazzi, L. L. Kosuda, L. H. Harnick, R. C. Brown, and N. R. Rose, Clin. Immunol. Immunopathol. 5, 182 (1976). s p. E. Bigazzi, L. L. Kosuda, K. C. Hsu, and G. A. Andres, J. Exp. Med. 143, 382 (1976). 9 p. E. Bigazzi, in "Vasectomy" (I. H. Lepow and R. Crozier, eds.), p. 339. Academic Press, New York, 1979. ~0p. E. Bigazzi, in "Reproductive Immunology" (N. Gleicher, ed.), p. 461. Liss, New York, 1981.
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tory to obtain and evaluate this animal model of organ-specific autoimmune disease. Vasectomy Materials
Rabbits, New Zealand White, male, 3.1-3.6 kg (Beckens Research Animal Farms, Sanborn, NY, or Jenks Rabbitry, Feeding Hills, MA) Acepromazine maleate (Aveco, Fort Dodge, IA) Ketamine hydrochloride (Ketaset, Bristol Laboratories, Syracuse, NY, or Ketalar, Parke-Davis, Morris Plains, NJ) Surgical instruments (scissors, knives, retractors, etc.), sterile Sutures (4-0 silk, chromic) (Ethicon Inc., Somerville, NJ) Furazolidone powder (Topazone, Norden, Smith Kline, Philadelphia, PA) Syringes, needles (22-gauge), centrifuge tubes, etc. Evaluation of Autoimmune Responses to Spermatozoa Materials
Cytocentrifuge Cytospin, SCA-0030 (Shandon Instruments, Sewickley, PA) Absolute methanol Phosphate-buffered saline (PBS), TM pH 7.0-7.5 Vasectomized and control rabbit sera, stored at - 70 ° Fluorescein isothiocyanate (FITC)-conjugated goat antibodies to rabbit immunoglobulins (available from commercial sources, e.g., Sigma Chemical Co., St. Louis, MO, or Cappel, Cooperbiomedical Inc., Malvern, PA) Fluorescence microscope (Leitz Dialux, equipped for transmitted light microscopy with an HBO 200-W mercury lamp, exciter filters BG12 and BG38, absorption filter K510) Tissue culture medium RPMI 1640 (Associated Biomedic Systems, Buffalo, NY) Buffered glycerol (1 volume PBS and 9 volumes glycerol) lo~Abbreviations: PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; CFA Freund's complete adjuvant; BUF, Buffalo; RT, rat thyroglobulin; BSA, bovine serum albumin; ART, antibodies to rat thyroglobulin; IIF, indirect immunofluorescence; ELISA, enzyme-linked immunosorbent assay; SAT, spontaneous autoimmune thyroiditis; EAT, experimentally induced thyroiditis.
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Immunohistopathology and Histopathology Materials Testes and kidneys from vasectomized and control rabbits (portions of specimens kept frozen at - 7 0 °, fixed in 10% buffered formalin, or fixed in a mixture of paraformaldehyde and glutaraldehyde) Cryostat (Wide Range Microtome-Cryostat WRC, Harris LoTemp Products, North Billerica, MA) Microtome Ultramicrotome (Reichert OMU2) FITC-conjugated goat antibodies to rabbit immunoglobulins (Sigma) FITC-conjugated goat antibodies to rabbit C3 (Cappel) Fluorescence microscope (Leitz Dialux with epiillumination) Light microscope (Leitz Dialux) Electron microscope (Philips EM 300) Buffered glycerol (1 volume PBS and 9 volumes glycerol) Methods
Vasectomy New Zealand White rabbits are bilaterally vasectomized by means of the following procedure. Food, but not water, is withheld for 12 hr prior to the administration of anesthesia. Rabbits weighing 3.1 -3.6 kg are tranquilized with a mixture of ketamine and acepromazine (1 ml acepromazine/ 10 mg ketamine) for a final dosage of 0.45 ml anesthetic per kg body weight. The anesthesized rabbit is secured to a board in a dorsally recumbent position, and the hair of the abdominal surface, inside of the hind legs, and around the penis and testes is carefully shaved. The area is prepared for surgery and draped. A vertical midline incision, about 2.5-cm long, is made in the skin of the suprapubic area (Fig. 1). With careful blunt dissection through this single mid-ventral incision, both spermatic cords are located and identified. The components of each cord are reached through a small cut in the cord wall, the vas deferens is carefully isolated, avoiding bleeding and trauma to the blood vessels, and ligated in two sites with silk sutures (Fig. 2). The part between the sutures, approximately 1 cm, is cut out (Fig. 3) and fixed in formalin for histological examination. The other components of the cord (arteries, veins, etc.), are gently pushed back into the cord, the small cut is sutured with silk, and the incision in the skin also sutured with silk. After surgery the rabbits receive furazolidone powder topically and a combination of penicillin and streptomycin (0.5 ml per rabbit) intramus-
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A
I
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FIG. i. Schematic outline of surgical incision for vasectomy in rabbits.
FIG. 2. Schematic drawing to demonstrate isolation and ligation of the vas deferens. Excision of the vas is performed along the dotted lines.
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FIG. 3. Schematic drawing of the vas deferens after ligation and excision.
cularly for 5 days. Bleedings are taken from the central artery of the ear approximately every 15- 30 days, and sera are stored at 70 ° until tested. Sham vasectomies are performed on rabbits of the same weight, anesthesized and treated following the procedure described above, with the exception that the vas deferens once isolated, is not ligated and cut out, but pushed back into the spermatic cord with the other cord components. Postsurgical treatment and trial bleedings are performed exactly as in the vasectomized group.
Detection of Antibodies to Spermatozoa by Indirect Immunofluorescence on Sperm Smears Spermatozoa are obtained from the epididymis of normal rabbits after unilateral or bilateral orchiectomy. After washing the spermatozoa in medium RPMI 1640, smears are prepared on clean microscope slides by using a cytocentrifuge at 1500 rpm for 10 min. Sperm smears are dried, fixed in absolute methanol for 30 min at room temperature, washed in PBS, and incubated for 30 min with 2-fold serial dilutions of rabbit serum starting with a dilution of 1 : 10. They are then washed in PBS for 30 min, incubated for 30 min with the FITC-conjugated immunoglobulin fraction of goat antiserum to rabbit immunoglobulins, and washed for 30 min. Sera obtained at different intervals from vasectomized rabbits (including a pre-
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vasectomy bleeding), sham vasectomized rabbits, and normal animals are tested in each experiment. As an additional control, the goat anti-rabbit immunoglobulin conjugate is also used on sperm smears without any previous incubation with serum. The slides are mounted in buffered glycerol, examined under a fluorescence microscope, and reactions are read as positive (subjectively rated as 1 +, 2 +, or 3 + according to the brightness), weakly positive ( + ) and negative.
Immunoh•topathology Small fragments of testes and kidneys obtained from sham vasectomized and control rabbits are snap-frozen in liquid nitrogen and stored at - 7 0 °. Four-micrometer-thick sections are cut in the cryostat and air dried. The sections are stained using FITC-conjugated antibodies to rabbit IgG, C3, albumin, and fibrinogen. The specificity of the conjugates is tested by immunodiffusion in agar and immunoelectrophoresis. Sections are incubated for 30 min with the appropriate dilution of the conjugate, washed for 30 min in PBS, and then mounted in buffered glycerol. The sections are examined under a fluorescence microscope.
Evaluation of Animal Model It can be expected that approximately 50% ofvasectomized rabbits will develop circulating antibodies against spermatozoa. In our laboratory, none of the prevasectomy sera and none of the sera from sham vasectomized and normal untreated rabbits have been found to give positive reactions. The kinetics of the antibody response to spermatozoal antigens should show that the percentage of positive animals increases with time. For example, we have found that 2% of sera from vasectomized rabbits are positive 2 and 4 months following vasectomy, 8.7% are positive at 6 months, and 27% at 8 months. In the following months the percentage of positive animals varies between 25 and 37%. Positive sera stain the acrosomal region of rabbit spermatozoa (as shown in Fig. 4), with titers of acrosomal antibodies ranging from l0 to 320. Rabbits with high and persistent levels of circulating antibodies to spermatozoa show thickening of basement membranes of seminiferous tubules and rare focal infiltrates of mononuclear cells in the interstitium. Occasionally, multinucleated spermatids, vacuolization of the basal portion of the tubules, as well as partial and total disappearance of Sertoli and spermatogonial cells may be observed. In thin sections, the main abnormalities of the basal lamina are small areas of increased contrasts, irregularities directed toward the epithelium, and accumulation of mononuclear
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FIo. 5. (a) Electron-opaquedeposits (arrowheads)within the basement membrane (asterisks) of a seminiferous tubule from a vasectomized rabbit. S, Spermatogonium; M, macrophage. The boxed areas are seen at higher magnificationin Fig. 5b, c. Original magnification X 7,000. (b, c) Three electron-opaque deposits (asterisks) between the split layers (arrowheads) of the lamina densa. Original magnification× 14,000. (d) Deposits of marked electron opacity in the basement membrane (asterisk) and within a spike (d). M indicates part of a macrophage. Original magnification× 14,000. Reproduced from Bigazziet al. 8
cells and rarely, polymorphonuclear leukocytes. By electron microscopy, the most characteristic finding is the deposition o f electron-opaque material in the basement membranes o f seminiferous tubules (Fig. 5). In rabbits with less severe or initial lesions, the deposits are found within the basement m e m b r a n e proper (lamina densa). In rabbits with more p r o n o u n c e d changes there is formation o f new layers o f epithelial basement m e m b r a n e and development o f "spikes" directed toward the epithelium. The electron-opaque deposits, frequently surrounded by newly formed layers o f epithelial basement membrane, are localized within the "spikes," between
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the collagenous layers of the basal lamina, or in the basement membranes of interstitial vessels in minimal amount. Granular deposits of rabbit IgG and C3 can be demonstrated along the seminiferous tubules by immunofluorescence (Fig. 6). In rabbits with mild or initial changes, the deposits are seen along the lamina densa that appears in proximity to the collagenous basal lamina. In contrast, in severely damaged tubules the immune deposits are separated from the collagenous basal lamina by a space containing macrophages. Spontaneous Autoimmune Thyroiditis As mentioned previously, there are several experimental models of autoimmune thyroiditis. The majority of these models are obtained by the injection of thyroid extract or purified thyroglobulin emulsified in Freund's complete adjuvant (CFA). 2 Other models are induced by the combination of thymectomy and irradiation of experimental animals or by the administration of chemicals such as methylcholanthrene.~In pure-bred obese strain (OS) chickens u and in rats of the Buffalo (BUF) inbred strain, however, autoimmune thyroiditis develops spontaneously, i.e., without any apparent cause or manipulation. Materials Buffalo (BUF) rats (Simonsen Laboratories, Gilroy, CA), retired female breeders, i.e., females older than 1 year that have produced a number of litters Rat thyroids, obtained at sacrifice of normal BUF rats or purchased from commercial sources (Pel-freeze Biologicals, Rogers, AK), kept frozen at - 70 ° ~25I (Amersham-Searle, Arlington Heights, IL) Lactoperoxidase (Calbiochem, La Jolla, CA) Sephadex G-75 (Pharmacia Fine Chemicals, Uppsala, Sweden) Gamma counter (Nuclear Chicago, Des Plaines, IL) FITC-conjugated rabbit antibodies to rat immunoglobulins (available from commercial sources, e.g., Cappel) Rabbit antibodies to rat immunoglobulins (Capped Fluorescence microscope (Leitz Dialux equipped with an HBO 200-W mercury lamp and set for transmitted light microscopy, exciter filters BGI2 and BG38, barrier filter K510) Buffered glycerol (1 volume PBS and 9 volumes glycerol) u G. Wick, J. Most, K. Schauenstein, G. Komer, H. Dietrich, A. Ziemiecki, R. Fassler, S. Schwarz, N. Neu, and K. Hala, Immunol. Today6, 359 (1985).
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Light microscope (Leitz Dialux) Electron microscope (Philips EM 300) Methods
Purification and Radiolabeling of Rat Thyroglobulin Rat thyroids are obtained from normal animals or purchased from commercial sources (Pel-freeze Biologicals). Thyroids are thawed, dissected from the trachea, cleaned from fat and connective tissue, and placed in a volume of PBS equal in milliliters to twice the weight of the tissue in grams. Tissues are ground in a Virtis homogenizer for 5 min. Centrifuge tubes containing the homogenate are placed overnight on wrist-action shaker. In the morning the thyroid homogenate is centrifuged for 30 min at 30,000 rpm in the No. 40 rotor of a Model L preparative ultracentrifuge (Beckman Instruments, Wakefield, MA). The pinkish supernatant, free of the dense pellet below and the lipid pellet above, is collected, the protein concentration determined by the biuret reaction, and the solution adjusted to 30 mg/ml. This material, which is designated "crude rat thyroid extract," is stored at 20 °. Rat thyroglobulin (RT) is prepared from "crude rat thyroid extract" by passing 10 ml of the thyroid extract through a Sephadex G-200 (Pharmacia) column. The column is washed with PBS, and fractions are collected automatically at 10-min intervals and monitored at 280 nm with a spectrophotometer. RT is usually recovered in the first protein peak eluting from the column, and it sediments as a single peak in the analytical ultracentrifuge with a sedimentation coefficient of approximately 19 S. Column-purified RT with a concentration of 1 mg/ml is stored at - 20 °. RT is labeled with '251 using lactoperoxidase, as previously described.'2 Briefly, 1 mCi (37 MBq) of '25I, lactoperoxidase (1% of the total protein), and 2/A of H202 are added to 100/~g of RT in 10/~1 of phosphate buffer, pH 7.3. The mixture is incubated for 15 min at room temperature, and the reaction stopped by addition of 2-mercaptoethanol. Next, 10/~1 of 50 m M KI is added, and the mixture incubated for 15 rain at room temperature. After the addition of 50/tl of bovine serum albumin (BSA) (40 mg/ml), the mixture is filtered through a Sephadex G-75 column. The efficiency of the radiolabeling procedure is controlled by precipitation with 20% trichloroacetic acid and determination of the radioactivity in the precipitate (usually, >90% of '251 is recovered in the pellet). Radiolabeled RT ('25I-RT) is aliquoted and stored at - 7 0 ° until use. 12 M. Zanetti and P. E. Bigazzi, Eur. J. Immunol. 11, 187 (1981).
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Detection of Circulating Antibodies to Rat Thyroglobulin Indirect Immunofluorescence. As previously described,13 sera are tested on rat thyroid cryostat sections, fixed in methanol at 56 ° for 3 min. Sera to be used are serially diluted in PBS, and dilutions are incubated on the sections for 30 min at room temperature in a humid chamber. Slides are rinsed, washed in PBS for 30 min, and then incubated with FITC-conjugated rabbit antisera to rat immunoglobulins for 30 min in a humid chamber. After an additional 30-min wash in PBS, sections are mounted in buffered glycerol and then examined under a fluorescence microscope. Sera containing antibodies to rat thyroglobulin (ART) give a floccular fluorescence of the thyroid colloid (Fig. 7). Reactions are scored on an arbitrary scale as 3 +, 2 +, 1 +, and - . Radioimmunoassay 12. In the first step of the radioimmunoassay, 25/tl of 125I-RT (10,000 cpm) is added to 25/tl of rat serum to be tested for ART, diluted 1 : 10 in 0.1% BSA in PBS. After an incubation of 30 min at 37 °, the total cpm in each tube is determined using a gamma counter. Following the incubation, rat IgG is precipitated by the addition of 400/ll of rabbit antiserum to rat IgG at the appropriate dilution (the concentration of antiserum necessary to precipitate the rat Ig in the mixture must be predetermined). After an additional incubation at 37 ° for 30 min, the tubes are centrifuged at 2,000 g for 220 min, washed with cold PBS, centrifuged again, and the cpm in the pellet counted. Tests are performed in duplicate, and the percentage of 125I-RT binding by the test serum is calculated from the following formula: 125I-RT Binding (%) --- cpm in the pellet × 100 total cpm
Histopathology Thyroids, stained with routine hematoxylin and eosin stains, are evaluated according to the percentage of infiltration of the organ. The condition of the whole thyroid is inferred from observation of six to eight serial sections taken from at least four separate cutting levels of the fixed tissue. In most cases both lobes are examined. The final evaluation is usually based on observations of both lobes. These determinations are necessarily quite subjective and qualitative; therefore, only three separate categories of thyroid pathology are made. Thyroids exhibiting definite thyroiditis, but with less than 30% of the parenchyma replaced by mononuclear cells, are said to have "low infiltration." Thyroids exhibiting 30-70% loss of normal 13 B. Noble, T. Yoshida, N. R. Rose, and P. E. Bigazzi, J. ImmunoL 117, 1447 (1976).
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structure are labeled "intermediate." When more than 70% of the thyroid is replaced by the infiltrating cells, the condition is described as "high infiltration." Evaluation of Animal Model On the basis of our experience, it can be expected that approximately 24% of female retired breeders (older than 1 year) will have circulating ART, detectable by indirect immunofluorescence (IIF)) 3 The presence of circulating ART, detected by IIF, correlates well with the incidence of inflammatory infiltration of the thyroid, present in 26% of the retired female breeders. Animals with low titers of serum ART detected by IIF have less infiltration of their thyroids, whereas rats with high antibody titers have intermediate levels of infiltration. On the other hand, rats with a high degree of infiltration have relatively low titers of ART by IIF. More recently, we have found that a similar correlation exists between circulating ART detected by radioimmunoassay and thyroiditis (Bigazzi el al., manuscript in preparation). In a group of 25 BUF rats, found positive for serum ART by radioimmunoassay, 96% had thyroiditis. On the other hand, 98% of another group of 52 BUF rats, negative for ART, had perfectly normal thyroids. Interestingly, similar studies, performed using an enzyme-linked immunosorbent assay (ELISA) to detect serum ART, 14 have failed to confirm this correlation between thyroiditis and circulating antibodies to thyroglobulin (Bigazzi et al., manuscript in preparation). The extent of thyroiditis may vary between affected animals from moderate (<30%) to intermediate (30-70%) and high (>70%) infiltration 13 (Fig. 8). The thyroid infiltrates observed in BUF rats are composed of lymphocytes, plasma cells, and macrophages, often with distinct germinal centers. No Hurthle cells are present. The incidence of spontaneous autoimmune thyroiditis (SAT) in untreated female BUF rats is a function of age and parity) No disease is found in female BUF rats less than l0 weeks of age, while 13% of rats aged 11 - 18 weeks show thyroid lesions. As previously mentioned, older female rats that have been bred repeatedly have a significantly higher incidence of thyroiditis than age-matched virgins. There is no difference between retired male breeders and nonbred males. In recent years, we have noticed a decrease in the incidence of thyroiditis in BUF female breeders (Bigazzi et aL, manuscript in preparation). This may be due to variations in environmental factors (especially viral and bacterial infections) or changes in the breeding practices of commercial suppliers. 14j. p. McCoy, Jr., J. H. Michaelson, and P. E. Bigazzi, LifeSci. 32, 109 (1983).
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Neonatal thymectomy increases the incidence of thyroiditis in BUF rats.' Thyroid infiltration is found in over 80% of these animals. The percentage of rats with thyroiditis seems to be correlated with the time of thymectomy, since the highest values are observed when thymectomy is performed within 24 hr of birth. When surgery is performed at 48 hr, the percentage of animals with thyroiditis decreases to 2 6 ° while thymectomies performed on 21-day-old BUF rats do not change the incidence of thyroiditis versus controls. Conclusions In this chapter we have reviewed two models of organ-specific autoimmune responses to spermatozoal antigens and a variety of immunohistotaneously. Both models can be very useful for investigations of the etiology and pathogenesis of organ-specific autoimmune disease. As an example, we have recently begun studies of the role of the idiotype-antiidiotype network in autoimmunity, utilizing BUF rats with SAT. 12,14-,6 The first model of organ-specific autoimmune disease has the advantage of being induced by a single surgical operation, vasectomy, without the need of any adjuvant. Interestingly, the findings in vasectomized rabbits resemble those of the experimental autoimmune orchitis obtained in rabbits by immunization with testicular antigens in CFA. 6 The autoimmune orchitis induced in rabbits by vasectomy is characterized by autoimmune responses to spermatozoal antigens and a variety of immunohistopathological and histopathological changes, with a picture very suggestive of in situ immune complex formation, a mechanism of organ-specific tissue damage previously observed at the level of the kidney or the thyroid in other experimental models of autoimmune disease. 8 The second model described is the spontaneous autoimmune thyroiditis of BUF rats. It should be noted first that one of the major advantages of this model is its occurrence in animals of an inbred strain. There are very few models of organ-specific autoimmune disease that arise spontaneously (i.e., without experimental manipulations) in inbred animals. Second, BUF rat thyroiditis is similar to the condition observed in humans because of its predilection for older females and the presence of circulating autoantibodies to thyroglobulin. The pathogenesis of SAT in BUF rats is still unclear. We have noted an absence of delayed-type hypersensitivity reactions in animals with SAT and a correlation between circulating autoantibodies to thyroglobulin and thyroid infiltration with mononuclear cells. is M. Zanetti, R. W. Barton, and P. E. Bigazzi, Cell. Immunol. 75, 292 (1983). 16 p. E. Bigazzi, Ann. N.Y. Acad. Sci. 475, 66(1986).
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These findings suggest that SAT of BUF rats may be autoantibody mediated in contrast to experimentally induced thyroiditis (EAT) that seems to be mediated by T cells. Neonatal thymectomy accelerates the onset and increases the incidence of SAT in BUF rats, while it abrogates EAT of rabbits, guinea pigs, rats, and mice, again suggesting that the experimentally induced disease has a different cellular basis from SAT. Acknowledgments These studies were supported by Grants HD17546 from NICHHD and ESO3684 from NIEHS, National Institutes of Health. We acknowledge the expert secretarial assistance of Ms. Ruth Conrod in the preparation of the manuscript.
[40] By PHILIP J.
Arthus Phenomenon BAILEY a n d DANIEL S. FLETCHER
Introduction The Arthus reaction is an inflammatory response resulting from the deposition of immune complexes within tissues. Tissue damage occurs due to the activation of components of the complement system, the infiltration of polymorphonuclear leukocytes (PMNs), 1 the release of lysosomal enzymes TMtogether with an increase in vascular permeability generated by the local release of mediators, and, in extreme cases, hemorrhage resulting from damage to the vessel wall. A study of the inflammatory response and the mechanism of tissue damage occurring in the Arthus phenomenon would therefore seem to have some relevance to the events occurring in a variety of pathological situations such as rheumatoid arthritis, glomerular nephritis, and systemic lupus erythematosus where immune complexes are deposited. The Arthus reaction can be elicited in a variety of animal species, rat, rabbit, and guinea pig being perhaps the most common, as well as in a number of different tissues including skin, the pleural space, synovial joints, and the paw. It can be induced basically in two ways: (1) as the J Abbreviations: PMNs, Polymorphonuclear leukocytes; RPA, reverse passive Arthus reaction; BSA, bovine serum albumin; R-ABSA, rabbit anti-BSA; PBS, phosphate-buffered saline; MPO, myeloperoxidase; HTAB, hexadecyltrimethylammonium bromide; HSA, human serum albumin. ~aC. G. Cochrane and B. S. Aiken, J. Exp. Med. 124, 733 (1966).
METHODS IN ENZYMOLOGY, VOL. 162
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TABLE III GLOMERULAR ANTIBODY BINDING (GAB) AND URINE PROTEIN EXCRETION 24 fir AFTER ADMINISTRATION OF SHEEP ANTI-RABBIT GBM TO NORMAL AND C6-DEFICIENT (C6D) RABmTSa
Groupb
GABc (gg IgG/104glomeruli)
Urine protein excretion (mg/g creatinine)
Normal (5)d C6D (6)
15.8 + 2.71e 18.7 + 1.99
42.0 + 26.Y 5.1 + 5.5
From Groggelet al. 44 b Rabbits are intravenously injected with sheep anti-rabbit GBM IgG (12 mg/kg) labeled with 125I. c Since kidneys are not perfused with saline, GAB is corrected for nonspecific binding by the double-isotope technique using nonimmune sheep 13'I-IgGas a plasma marker. a Number of rabbits studied is given in parentheses. e Values are means + SD.
a
development o f heterologous-phase proteinuria. 44 Urine protein excretion is measured for 24 hr after antibody injection, and glomerular binding is assessed by the dual-isotope m e t h o d at the end o f the urine collection period. The results show a lower rate o f urine protein excretion in the C6-deficient group, which cannot be attributed to their having less glomerular antibody. Acknowledgments This work was supported by U.S. Public Health Service Grant RO1 DK30932 from the National Institute of Diabetes, Digestive and Kidney Diseases, by an American Heart Association Established Investigator Award (D.J,S.), and by the Kidney Foundation of Canada and
Medical Research Council of Canada (A.V.C.).
[39] Autoimmune Orchitis and Thyroiditis By PIERLUIGI
E. BIGAZZI
Introduction Studies o f organ-specific a u t o i m m u n e diseases occurring in laboratory animals have provided useful and often illuminating information regarding the etiologic and pathogenetic mechanisms o f h u m a n a u t o i m m u n e disease. A u t o i m m u n e disease can be induced in animals by the administraMETHODS IN ENZYMOLOGY, VOL. 162
Copyright© 1988by AcademicPress,Inc. All fightsof reproductionin any form~erved.
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tion of drugs and other chemicals 1 or X-irradiation and by surgical manipulations (e.g., thymectomy). More commonly, such experimental models of autoimmunity are obtained by the immunization of laboratory animals with homologous or heterologous organ-specific antigens, usually emulsified in Freund's complete adjuvant or in association with other adjuvants (e.g., Bordetella pertussis). 2,3 However, as Burnet once noted, "An elderly woman with Hashimoto's disease of the thyroid has not been injected with rabbit thyroid in Freund's complete adjuvant." 4 In other words, there is a need for animal models of autoimmune disease that are not dependent on the artificial administration of antigens and adjuvants and that resemble more closely the situation occurring in humans. For this reason, we have suggested that careful investigation of spontaneous organ-specific autoimmune disease of animals may provide additional insights into human iUness. 5 In this chapter, we review both an experimentally induced and a spontaneous model of organ-specific autoimmune disease. Postvasectomy Autoimmune Orchitis Autoimmune orchitis is induced experimentally in a variety of animal species by immunization with spermatozoa or testicular antigens, emulsified with Freund's complete adjuvant or associated with B. pertussis. 6 However, there is one model of autoimmune orchitis occurring in rabbits, initiated by occlusive surgery of the vas deferens (vasectomy) and not dependent on the administration of exogenous adjuvants. 7-1° In the following section we describe in detail the procedures employed in our laboraP. E. Bigazzi, in "Immunotoxicology and Immunopharmacology" (J. Dean, M. I. Luster, A. E. Munson, and H. Amos, ¢ds.), p. 277. Raven, New York, 1985. 2 p. E. Bigazzi and N. R. Rose, in "The Autoimmune Diseases" (N. R. Rose and I. R. Mackay, eds.), p. 161. Academic Press, Orlando, Florida, 1985. 3 p. E. Bigazzi, in "Autoimmunity and Endocrine Disease" (R. Volp¢, ed.), p 345. Dekker, New York, 1985. 4 F. M. Burnet, "Auto-immunity and Auto-immune Disease," p. 101. Davis, Philadelphia, Pennsylvania, 1972. s p. E. Bigazzi and N. R. Rose, Prog. Allergy 19, 245 (1975). 6 K. S. K. Tung and A. C. Menge, in "The Antoimmune Diseases" (N. R. Rose and I. R. Mackay, eds.), p 537. Academic Press, Orlando, Florida, 1985. 7 p. E. Bigazzi, L. L. Kosuda, L. H. Harnick, R. C. Brown, and N. R. Rose, Clin. Immunol. Immunopathol. 5, 182 (1976). s p. E. Bigazzi, L. L. Kosuda, K. C. Hsu, and G. A. Andres, J. Exp. Med. 143, 382 (1976). 9 p. E. Bigazzi, in "Vasectomy" (I. H. Lepow and R. Crozier, eds.), p. 339. Academic Press, New York, 1979. ~0p. E. Bigazzi, in "Reproductive Immunology" (N. Gleicher, ed.), p. 461. Liss, New York, 1981.
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tory to obtain and evaluate this animal model of organ-specific autoimmune disease. Vasectomy Materials
Rabbits, New Zealand White, male, 3.1-3.6 kg (Beckens Research Animal Farms, Sanborn, NY, or Jenks Rabbitry, Feeding Hills, MA) Acepromazine maleate (Aveco, Fort Dodge, IA) Ketamine hydrochloride (Ketaset, Bristol Laboratories, Syracuse, NY, or Ketalar, Parke-Davis, Morris Plains, NJ) Surgical instruments (scissors, knives, retractors, etc.), sterile Sutures (4-0 silk, chromic) (Ethicon Inc., Somerville, NJ) Furazolidone powder (Topazone, Norden, Smith Kline, Philadelphia, PA) Syringes, needles (22-gauge), centrifuge tubes, etc. Evaluation of Autoimmune Responses to Spermatozoa Materials
Cytocentrifuge Cytospin, SCA-0030 (Shandon Instruments, Sewickley, PA) Absolute methanol Phosphate-buffered saline (PBS), TM pH 7.0-7.5 Vasectomized and control rabbit sera, stored at - 70 ° Fluorescein isothiocyanate (FITC)-conjugated goat antibodies to rabbit immunoglobulins (available from commercial sources, e.g., Sigma Chemical Co., St. Louis, MO, or Cappel, Cooperbiomedical Inc., Malvern, PA) Fluorescence microscope (Leitz Dialux, equipped for transmitted light microscopy with an HBO 200-W mercury lamp, exciter filters BG12 and BG38, absorption filter K510) Tissue culture medium RPMI 1640 (Associated Biomedic Systems, Buffalo, NY) Buffered glycerol (1 volume PBS and 9 volumes glycerol) lo~Abbreviations: PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; CFA Freund's complete adjuvant; BUF, Buffalo; RT, rat thyroglobulin; BSA, bovine serum albumin; ART, antibodies to rat thyroglobulin; IIF, indirect immunofluorescence; ELISA, enzyme-linked immunosorbent assay; SAT, spontaneous autoimmune thyroiditis; EAT, experimentally induced thyroiditis.
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Immunohistopathology and Histopathology Materials Testes and kidneys from vasectomized and control rabbits (portions of specimens kept frozen at - 7 0 °, fixed in 10% buffered formalin, or fixed in a mixture of paraformaldehyde and glutaraldehyde) Cryostat (Wide Range Microtome-Cryostat WRC, Harris LoTemp Products, North Billerica, MA) Microtome Ultramicrotome (Reichert OMU2) FITC-conjugated goat antibodies to rabbit immunoglobulins (Sigma) FITC-conjugated goat antibodies to rabbit C3 (Cappel) Fluorescence microscope (Leitz Dialux with epiillumination) Light microscope (Leitz Dialux) Electron microscope (Philips EM 300) Buffered glycerol (1 volume PBS and 9 volumes glycerol) Methods
Vasectomy New Zealand White rabbits are bilaterally vasectomized by means of the following procedure. Food, but not water, is withheld for 12 hr prior to the administration of anesthesia. Rabbits weighing 3.1 -3.6 kg are tranquilized with a mixture of ketamine and acepromazine (1 ml acepromazine/ 10 mg ketamine) for a final dosage of 0.45 ml anesthetic per kg body weight. The anesthesized rabbit is secured to a board in a dorsally recumbent position, and the hair of the abdominal surface, inside of the hind legs, and around the penis and testes is carefully shaved. The area is prepared for surgery and draped. A vertical midline incision, about 2.5-cm long, is made in the skin of the suprapubic area (Fig. 1). With careful blunt dissection through this single mid-ventral incision, both spermatic cords are located and identified. The components of each cord are reached through a small cut in the cord wall, the vas deferens is carefully isolated, avoiding bleeding and trauma to the blood vessels, and ligated in two sites with silk sutures (Fig. 2). The part between the sutures, approximately 1 cm, is cut out (Fig. 3) and fixed in formalin for histological examination. The other components of the cord (arteries, veins, etc.), are gently pushed back into the cord, the small cut is sutured with silk, and the incision in the skin also sutured with silk. After surgery the rabbits receive furazolidone powder topically and a combination of penicillin and streptomycin (0.5 ml per rabbit) intramus-
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FIG. i. Schematic outline of surgical incision for vasectomy in rabbits.
FIG. 2. Schematic drawing to demonstrate isolation and ligation of the vas deferens. Excision of the vas is performed along the dotted lines.
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FIG. 3. Schematic drawing of the vas deferens after ligation and excision.
cularly for 5 days. Bleedings are taken from the central artery of the ear approximately every 15- 30 days, and sera are stored at 70 ° until tested. Sham vasectomies are performed on rabbits of the same weight, anesthesized and treated following the procedure described above, with the exception that the vas deferens once isolated, is not ligated and cut out, but pushed back into the spermatic cord with the other cord components. Postsurgical treatment and trial bleedings are performed exactly as in the vasectomized group.
Detection of Antibodies to Spermatozoa by Indirect Immunofluorescence on Sperm Smears Spermatozoa are obtained from the epididymis of normal rabbits after unilateral or bilateral orchiectomy. After washing the spermatozoa in medium RPMI 1640, smears are prepared on clean microscope slides by using a cytocentrifuge at 1500 rpm for 10 min. Sperm smears are dried, fixed in absolute methanol for 30 min at room temperature, washed in PBS, and incubated for 30 min with 2-fold serial dilutions of rabbit serum starting with a dilution of 1 : 10. They are then washed in PBS for 30 min, incubated for 30 min with the FITC-conjugated immunoglobulin fraction of goat antiserum to rabbit immunoglobulins, and washed for 30 min. Sera obtained at different intervals from vasectomized rabbits (including a pre-
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vasectomy bleeding), sham vasectomized rabbits, and normal animals are tested in each experiment. As an additional control, the goat anti-rabbit immunoglobulin conjugate is also used on sperm smears without any previous incubation with serum. The slides are mounted in buffered glycerol, examined under a fluorescence microscope, and reactions are read as positive (subjectively rated as 1 +, 2 +, or 3 + according to the brightness), weakly positive ( + ) and negative.
Immunoh•topathology Small fragments of testes and kidneys obtained from sham vasectomized and control rabbits are snap-frozen in liquid nitrogen and stored at - 7 0 °. Four-micrometer-thick sections are cut in the cryostat and air dried. The sections are stained using FITC-conjugated antibodies to rabbit IgG, C3, albumin, and fibrinogen. The specificity of the conjugates is tested by immunodiffusion in agar and immunoelectrophoresis. Sections are incubated for 30 min with the appropriate dilution of the conjugate, washed for 30 min in PBS, and then mounted in buffered glycerol. The sections are examined under a fluorescence microscope.
Evaluation of Animal Model It can be expected that approximately 50% ofvasectomized rabbits will develop circulating antibodies against spermatozoa. In our laboratory, none of the prevasectomy sera and none of the sera from sham vasectomized and normal untreated rabbits have been found to give positive reactions. The kinetics of the antibody response to spermatozoal antigens should show that the percentage of positive animals increases with time. For example, we have found that 2% of sera from vasectomized rabbits are positive 2 and 4 months following vasectomy, 8.7% are positive at 6 months, and 27% at 8 months. In the following months the percentage of positive animals varies between 25 and 37%. Positive sera stain the acrosomal region of rabbit spermatozoa (as shown in Fig. 4), with titers of acrosomal antibodies ranging from l0 to 320. Rabbits with high and persistent levels of circulating antibodies to spermatozoa show thickening of basement membranes of seminiferous tubules and rare focal infiltrates of mononuclear cells in the interstitium. Occasionally, multinucleated spermatids, vacuolization of the basal portion of the tubules, as well as partial and total disappearance of Sertoli and spermatogonial cells may be observed. In thin sections, the main abnormalities of the basal lamina are small areas of increased contrasts, irregularities directed toward the epithelium, and accumulation of mononuclear
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FIo. 5. (a) Electron-opaquedeposits (arrowheads)within the basement membrane (asterisks) of a seminiferous tubule from a vasectomized rabbit. S, Spermatogonium; M, macrophage. The boxed areas are seen at higher magnificationin Fig. 5b, c. Original magnification X 7,000. (b, c) Three electron-opaque deposits (asterisks) between the split layers (arrowheads) of the lamina densa. Original magnification× 14,000. (d) Deposits of marked electron opacity in the basement membrane (asterisk) and within a spike (d). M indicates part of a macrophage. Original magnification× 14,000. Reproduced from Bigazziet al. 8
cells and rarely, polymorphonuclear leukocytes. By electron microscopy, the most characteristic finding is the deposition o f electron-opaque material in the basement membranes o f seminiferous tubules (Fig. 5). In rabbits with less severe or initial lesions, the deposits are found within the basement m e m b r a n e proper (lamina densa). In rabbits with more p r o n o u n c e d changes there is formation o f new layers o f epithelial basement m e m b r a n e and development o f "spikes" directed toward the epithelium. The electron-opaque deposits, frequently surrounded by newly formed layers o f epithelial basement membrane, are localized within the "spikes," between
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the collagenous layers of the basal lamina, or in the basement membranes of interstitial vessels in minimal amount. Granular deposits of rabbit IgG and C3 can be demonstrated along the seminiferous tubules by immunofluorescence (Fig. 6). In rabbits with mild or initial changes, the deposits are seen along the lamina densa that appears in proximity to the collagenous basal lamina. In contrast, in severely damaged tubules the immune deposits are separated from the collagenous basal lamina by a space containing macrophages. Spontaneous Autoimmune Thyroiditis As mentioned previously, there are several experimental models of autoimmune thyroiditis. The majority of these models are obtained by the injection of thyroid extract or purified thyroglobulin emulsified in Freund's complete adjuvant (CFA). 2 Other models are induced by the combination of thymectomy and irradiation of experimental animals or by the administration of chemicals such as methylcholanthrene.~In pure-bred obese strain (OS) chickens u and in rats of the Buffalo (BUF) inbred strain, however, autoimmune thyroiditis develops spontaneously, i.e., without any apparent cause or manipulation. Materials Buffalo (BUF) rats (Simonsen Laboratories, Gilroy, CA), retired female breeders, i.e., females older than 1 year that have produced a number of litters Rat thyroids, obtained at sacrifice of normal BUF rats or purchased from commercial sources (Pel-freeze Biologicals, Rogers, AK), kept frozen at - 70 ° ~25I (Amersham-Searle, Arlington Heights, IL) Lactoperoxidase (Calbiochem, La Jolla, CA) Sephadex G-75 (Pharmacia Fine Chemicals, Uppsala, Sweden) Gamma counter (Nuclear Chicago, Des Plaines, IL) FITC-conjugated rabbit antibodies to rat immunoglobulins (available from commercial sources, e.g., Cappel) Rabbit antibodies to rat immunoglobulins (Capped Fluorescence microscope (Leitz Dialux equipped with an HBO 200-W mercury lamp and set for transmitted light microscopy, exciter filters BGI2 and BG38, barrier filter K510) Buffered glycerol (1 volume PBS and 9 volumes glycerol) u G. Wick, J. Most, K. Schauenstein, G. Komer, H. Dietrich, A. Ziemiecki, R. Fassler, S. Schwarz, N. Neu, and K. Hala, Immunol. Today6, 359 (1985).
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Light microscope (Leitz Dialux) Electron microscope (Philips EM 300) Methods
Purification and Radiolabeling of Rat Thyroglobulin Rat thyroids are obtained from normal animals or purchased from commercial sources (Pel-freeze Biologicals). Thyroids are thawed, dissected from the trachea, cleaned from fat and connective tissue, and placed in a volume of PBS equal in milliliters to twice the weight of the tissue in grams. Tissues are ground in a Virtis homogenizer for 5 min. Centrifuge tubes containing the homogenate are placed overnight on wrist-action shaker. In the morning the thyroid homogenate is centrifuged for 30 min at 30,000 rpm in the No. 40 rotor of a Model L preparative ultracentrifuge (Beckman Instruments, Wakefield, MA). The pinkish supernatant, free of the dense pellet below and the lipid pellet above, is collected, the protein concentration determined by the biuret reaction, and the solution adjusted to 30 mg/ml. This material, which is designated "crude rat thyroid extract," is stored at 20 °. Rat thyroglobulin (RT) is prepared from "crude rat thyroid extract" by passing 10 ml of the thyroid extract through a Sephadex G-200 (Pharmacia) column. The column is washed with PBS, and fractions are collected automatically at 10-min intervals and monitored at 280 nm with a spectrophotometer. RT is usually recovered in the first protein peak eluting from the column, and it sediments as a single peak in the analytical ultracentrifuge with a sedimentation coefficient of approximately 19 S. Column-purified RT with a concentration of 1 mg/ml is stored at - 20 °. RT is labeled with '251 using lactoperoxidase, as previously described.'2 Briefly, 1 mCi (37 MBq) of '25I, lactoperoxidase (1% of the total protein), and 2/A of H202 are added to 100/~g of RT in 10/~1 of phosphate buffer, pH 7.3. The mixture is incubated for 15 min at room temperature, and the reaction stopped by addition of 2-mercaptoethanol. Next, 10/~1 of 50 m M KI is added, and the mixture incubated for 15 rain at room temperature. After the addition of 50/tl of bovine serum albumin (BSA) (40 mg/ml), the mixture is filtered through a Sephadex G-75 column. The efficiency of the radiolabeling procedure is controlled by precipitation with 20% trichloroacetic acid and determination of the radioactivity in the precipitate (usually, >90% of '251 is recovered in the pellet). Radiolabeled RT ('25I-RT) is aliquoted and stored at - 7 0 ° until use. 12 M. Zanetti and P. E. Bigazzi, Eur. J. Immunol. 11, 187 (1981).
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Detection of Circulating Antibodies to Rat Thyroglobulin Indirect Immunofluorescence. As previously described,13 sera are tested on rat thyroid cryostat sections, fixed in methanol at 56 ° for 3 min. Sera to be used are serially diluted in PBS, and dilutions are incubated on the sections for 30 min at room temperature in a humid chamber. Slides are rinsed, washed in PBS for 30 min, and then incubated with FITC-conjugated rabbit antisera to rat immunoglobulins for 30 min in a humid chamber. After an additional 30-min wash in PBS, sections are mounted in buffered glycerol and then examined under a fluorescence microscope. Sera containing antibodies to rat thyroglobulin (ART) give a floccular fluorescence of the thyroid colloid (Fig. 7). Reactions are scored on an arbitrary scale as 3 +, 2 +, 1 +, and - . Radioimmunoassay 12. In the first step of the radioimmunoassay, 25/tl of 125I-RT (10,000 cpm) is added to 25/tl of rat serum to be tested for ART, diluted 1 : 10 in 0.1% BSA in PBS. After an incubation of 30 min at 37 °, the total cpm in each tube is determined using a gamma counter. Following the incubation, rat IgG is precipitated by the addition of 400/ll of rabbit antiserum to rat IgG at the appropriate dilution (the concentration of antiserum necessary to precipitate the rat Ig in the mixture must be predetermined). After an additional incubation at 37 ° for 30 min, the tubes are centrifuged at 2,000 g for 220 min, washed with cold PBS, centrifuged again, and the cpm in the pellet counted. Tests are performed in duplicate, and the percentage of 125I-RT binding by the test serum is calculated from the following formula: 125I-RT Binding (%) --- cpm in the pellet × 100 total cpm
Histopathology Thyroids, stained with routine hematoxylin and eosin stains, are evaluated according to the percentage of infiltration of the organ. The condition of the whole thyroid is inferred from observation of six to eight serial sections taken from at least four separate cutting levels of the fixed tissue. In most cases both lobes are examined. The final evaluation is usually based on observations of both lobes. These determinations are necessarily quite subjective and qualitative; therefore, only three separate categories of thyroid pathology are made. Thyroids exhibiting definite thyroiditis, but with less than 30% of the parenchyma replaced by mononuclear cells, are said to have "low infiltration." Thyroids exhibiting 30-70% loss of normal 13 B. Noble, T. Yoshida, N. R. Rose, and P. E. Bigazzi, J. ImmunoL 117, 1447 (1976).
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structure are labeled "intermediate." When more than 70% of the thyroid is replaced by the infiltrating cells, the condition is described as "high infiltration." Evaluation of Animal Model On the basis of our experience, it can be expected that approximately 24% of female retired breeders (older than 1 year) will have circulating ART, detectable by indirect immunofluorescence (IIF)) 3 The presence of circulating ART, detected by IIF, correlates well with the incidence of inflammatory infiltration of the thyroid, present in 26% of the retired female breeders. Animals with low titers of serum ART detected by IIF have less infiltration of their thyroids, whereas rats with high antibody titers have intermediate levels of infiltration. On the other hand, rats with a high degree of infiltration have relatively low titers of ART by IIF. More recently, we have found that a similar correlation exists between circulating ART detected by radioimmunoassay and thyroiditis (Bigazzi el al., manuscript in preparation). In a group of 25 BUF rats, found positive for serum ART by radioimmunoassay, 96% had thyroiditis. On the other hand, 98% of another group of 52 BUF rats, negative for ART, had perfectly normal thyroids. Interestingly, similar studies, performed using an enzyme-linked immunosorbent assay (ELISA) to detect serum ART, 14 have failed to confirm this correlation between thyroiditis and circulating antibodies to thyroglobulin (Bigazzi et al., manuscript in preparation). The extent of thyroiditis may vary between affected animals from moderate (<30%) to intermediate (30-70%) and high (>70%) infiltration 13 (Fig. 8). The thyroid infiltrates observed in BUF rats are composed of lymphocytes, plasma cells, and macrophages, often with distinct germinal centers. No Hurthle cells are present. The incidence of spontaneous autoimmune thyroiditis (SAT) in untreated female BUF rats is a function of age and parity) No disease is found in female BUF rats less than l0 weeks of age, while 13% of rats aged 11 - 18 weeks show thyroid lesions. As previously mentioned, older female rats that have been bred repeatedly have a significantly higher incidence of thyroiditis than age-matched virgins. There is no difference between retired male breeders and nonbred males. In recent years, we have noticed a decrease in the incidence of thyroiditis in BUF female breeders (Bigazzi et aL, manuscript in preparation). This may be due to variations in environmental factors (especially viral and bacterial infections) or changes in the breeding practices of commercial suppliers. 14j. p. McCoy, Jr., J. H. Michaelson, and P. E. Bigazzi, LifeSci. 32, 109 (1983).
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Neonatal thymectomy increases the incidence of thyroiditis in BUF rats.' Thyroid infiltration is found in over 80% of these animals. The percentage of rats with thyroiditis seems to be correlated with the time of thymectomy, since the highest values are observed when thymectomy is performed within 24 hr of birth. When surgery is performed at 48 hr, the percentage of animals with thyroiditis decreases to 2 6 ° while thymectomies performed on 21-day-old BUF rats do not change the incidence of thyroiditis versus controls. Conclusions In this chapter we have reviewed two models of organ-specific autoimmune responses to spermatozoal antigens and a variety of immunohistotaneously. Both models can be very useful for investigations of the etiology and pathogenesis of organ-specific autoimmune disease. As an example, we have recently begun studies of the role of the idiotype-antiidiotype network in autoimmunity, utilizing BUF rats with SAT. 12,14-,6 The first model of organ-specific autoimmune disease has the advantage of being induced by a single surgical operation, vasectomy, without the need of any adjuvant. Interestingly, the findings in vasectomized rabbits resemble those of the experimental autoimmune orchitis obtained in rabbits by immunization with testicular antigens in CFA. 6 The autoimmune orchitis induced in rabbits by vasectomy is characterized by autoimmune responses to spermatozoal antigens and a variety of immunohistopathological and histopathological changes, with a picture very suggestive of in situ immune complex formation, a mechanism of organ-specific tissue damage previously observed at the level of the kidney or the thyroid in other experimental models of autoimmune disease. 8 The second model described is the spontaneous autoimmune thyroiditis of BUF rats. It should be noted first that one of the major advantages of this model is its occurrence in animals of an inbred strain. There are very few models of organ-specific autoimmune disease that arise spontaneously (i.e., without experimental manipulations) in inbred animals. Second, BUF rat thyroiditis is similar to the condition observed in humans because of its predilection for older females and the presence of circulating autoantibodies to thyroglobulin. The pathogenesis of SAT in BUF rats is still unclear. We have noted an absence of delayed-type hypersensitivity reactions in animals with SAT and a correlation between circulating autoantibodies to thyroglobulin and thyroid infiltration with mononuclear cells. is M. Zanetti, R. W. Barton, and P. E. Bigazzi, Cell. Immunol. 75, 292 (1983). 16 p. E. Bigazzi, Ann. N.Y. Acad. Sci. 475, 66(1986).
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These findings suggest that SAT of BUF rats may be autoantibody mediated in contrast to experimentally induced thyroiditis (EAT) that seems to be mediated by T cells. Neonatal thymectomy accelerates the onset and increases the incidence of SAT in BUF rats, while it abrogates EAT of rabbits, guinea pigs, rats, and mice, again suggesting that the experimentally induced disease has a different cellular basis from SAT. Acknowledgments These studies were supported by Grants HD17546 from NICHHD and ESO3684 from NIEHS, National Institutes of Health. We acknowledge the expert secretarial assistance of Ms. Ruth Conrod in the preparation of the manuscript.
[40] By PHILIP J.
Arthus Phenomenon BAILEY a n d DANIEL S. FLETCHER
Introduction The Arthus reaction is an inflammatory response resulting from the deposition of immune complexes within tissues. Tissue damage occurs due to the activation of components of the complement system, the infiltration of polymorphonuclear leukocytes (PMNs), 1 the release of lysosomal enzymes TMtogether with an increase in vascular permeability generated by the local release of mediators, and, in extreme cases, hemorrhage resulting from damage to the vessel wall. A study of the inflammatory response and the mechanism of tissue damage occurring in the Arthus phenomenon would therefore seem to have some relevance to the events occurring in a variety of pathological situations such as rheumatoid arthritis, glomerular nephritis, and systemic lupus erythematosus where immune complexes are deposited. The Arthus reaction can be elicited in a variety of animal species, rat, rabbit, and guinea pig being perhaps the most common, as well as in a number of different tissues including skin, the pleural space, synovial joints, and the paw. It can be induced basically in two ways: (1) as the J Abbreviations: PMNs, Polymorphonuclear leukocytes; RPA, reverse passive Arthus reaction; BSA, bovine serum albumin; R-ABSA, rabbit anti-BSA; PBS, phosphate-buffered saline; MPO, myeloperoxidase; HTAB, hexadecyltrimethylammonium bromide; HSA, human serum albumin. ~aC. G. Cochrane and B. S. Aiken, J. Exp. Med. 124, 733 (1966).
METHODS IN ENZYMOLOGY, VOL. 162
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