The Thyroid Cytotoxic Autoantibody

THE THYROID CYTOTOXIC AUTOANTIBODY

I. J. Forbes, … , Deborah Doniach, I. L. Solomon

J Clin Invest. 1962;41(5):996-1006. https://doi.org/10.1172/JCI104579.

Research Article

Find the latest version: https://jci.me/104579/pdf Journal of Clinical Investigation Vol. 41, No. 5, 1962 THE THYROID CYTOTOXIC AUTOANTIBODY By I. J. FORBES,* I. M. ROITT, DEBORAH DONIACH AND I. L. SOLOMON t (From the Middlesex Hospital Medical School, London, England) (Submitted for publication November 27, 1961; accepted January 18, 1962)

The presence of circulating autoantibodies in micromethod of Donnelley (6), and was known to sup- patients with Hashimoto's disease suggests that port good cell growth. Fresh normal serum was always used in tests with serum fractions or absorbed sera. autoimmunity is implicated in the disease process, Guinea pig serum could be used as a source of comple- but the ultimate proof of an autoimmune patho- ment but was occasionally cytotoxic. The cultures were genesis of thyroiditis and other human diseases incubated for 18 to 24 hours at 370C. must be obtained by demonstrating the autoag- The sensitivity of each gland was established by in- gressive action of antibodies or immunologically cluding as controls known weakly cytotoxic sera and competent cells on the living tissue in its normal potent Hashimoto sera, sometimes set up in dilutions. With sensitive glands, all the cells were killed by a environment. strong standard serum; the weaker sera usually allowed The demonstration by Pulvertaft, Doniach, Roitt a few clumps of cells to be established. Where cells and Hudson (1, 2) of a serum factor capable of survived in final dilutions of less than 1: 120 of the destroying human thyroid cells in tissue culture standard strong serum, the gland was too insensitive to is an advance in this direction. This finding has permit evaluation of unknown sera. Provided the gland was sensitive, a serum was judged to be cytotoxic when been confirmed and amplified by Irvine (3, 4) and either no clumps of healthy cells were visible or there by Goudie and McCallum (5). Further investi- was a gross reduction of surviving cells relative to con- gations of this phenomenon are reported in the trol cultures. All positive sera were retested in at least present paper. The cytotoxic factor is shown to one subsequent culture. be an the micro- Zone electrophoresis on "Pevikon" blocks. This was antibody reacting with thyroid carried out by a method similar to that of Muller- somal antigen; its mode of action has been further Eberhard (7). Approximately 50 g of polyvinyl-chlo- studied and its incidence determined in a variety ride powder (Pevikon, Fosfatbolaget, Stockviksverkin, of thyroid conditions and in persons without Sweden) washed three times in barbiturate buffer, pH overt thyroid disease. 8.6, was made into a thick slurry and poured into a Perspex trough, 7 X 2 X 1/2 inches. The serum was dialyzed overnight at 40C against the buffer and a trace METHODS AND MATERIALS of bromophenol blue added. After equilibration of the Tissue culture. Primary monolayer cultures of trypsin- block in the electrode chamber at 4VC for 30 minutes, 1.5 dispersed thyroid epithelium were set up in chambers ml of serum was added to a depression in the block, 1 prepared on microscope slides by the method of Pulver- inch from the cathodal end. Electrophoresis was con- taft and co-workers (1). The chambers consisted of a tinued for approximately 18 hours in the cold, using a plastic ring attached to a histology slide with silicone current of 7 ma until the blue-stained albumin was grease, and sealed with a coverslip. Fresh thyroid tis- within 1 inch of the anodal end of the block. At the end sue was dispersed for 1 hour in 0.25 per cent trypsin of electrophoresis the block was cut transversely into (Difco, 1: 250), and after centrifugation a suitable six equal portions. The fractions were stirred with 2 inoculum was prepared by adding a concentrated sus- ml of Parker's 199 medium in tubes and the supernatant pension of cells to Parker 199 medium until a density of recovered after centrifugation. Each fraction was dia- 5 epithelial clumps per field was seen when a drop was lyzed against 250 ml of phosphate-buffered saline at 4°C viewed through a 1oX objective. The ideal cell con- (8) overnight, then against 10 ml of Parker 199 for centration resulted in the growth of 30 to 50 cell clumps 8 hours. The fractions were stored at - 20°C until in a control chamber. Each chamber contained 0.1 ml tested. of test serum, 0.1 ml fresh normal serum, and 0.6 ml of The protein fractions were identified by immunoelec- cell inoculum. The fresh normal serum used routinely trophoresis, with Scheidegger's (9) micromethod. Four had a complement titer of between 160 and 320 minimal drops of each fraction in serial fivefold dilutions were hemolytic doses (MHD) per ml, as determined by the set up for tissue culture; 0.2 ml of fresh normal human serum was added to each dilution. A duplicate undiluted * Present address: University of Michigan Medical tube from each fraction was inactivated at 56°C for 30 Center, Ann Arbor, Mich. minutes before culture to ensure that cytotoxicity was t Present address: Children's Hospital, Columbus, Ohio. complement dependent. 996 THYROID CYTOTOXIC AUTO-ANTIBODY 997 Sodium sulfate precipitation of 'y-globulins from cy- thyroid gland, as described by Holborow, Brown, Roitt totoxic sera. Two vol of 27 per cent sodium sulfate in and Doniach (11). Antibodies to the colloid were dem- phosphate buffer, pH 7.8, was added to 1 vol of a cyto- onstrated by alcohol-fixed thyroid sections (12). The toxic serum, over a period of 10 minutes, with continu- tanned cell hemagglutination (TRC) test for thyroglobu- ous stirring. After centrifugation at 10,000 rpm for 10 lin antibodies was carried out with a formalinized sheep minutes at room temperature, the supernatant was de- cell preparation (13). The complement fixation test canted and saved. The precipitate was washed in 18 (CFT) was done with thyrotoxic gland homogenate using per cent sodium sulfate and after centrifugation was dis- 2 MHD (14) or 11/4 MHD of complement. solved in a volume of distilled water equal to the volume Enzymes. The following were used for cell dispersal: of the supernatant. The solutions were dialyzed against trypsin (1: 250 Difco Lab., Detroit, Mich.); collagenase phosphate-buffered saline and Parker's 199 medium for (Worthington Biochemicals Corp., Freehold, N. J.); use in the tissue culture test and for the detection of cy- papain (British Drug Houses, Poole, England); and ficin toplasmic antibodies by Coons' technique. (crude, Light & Co., Colnbrook, England). Chromatography of -y-globulin obtained by zone elec- Case material. Patients with primary myxedema and trophoresis on Pezikon blocks. The first three fractions thyrotoxicosis were accepted if the clinical evidence was containing y-globulin were used from two Pevikon blocks unequivocal or was confirmed by laboratory studies. prepared according to the method described above. Cases of nontoxic nodular goiter may have included some Chromatography was carried out on a diethylaminoethyl in which focal thyroiditis was present but was not (DEAE)-cellulose column by the method of Fahey and disclosed by laboratory studies. The patients with Hashi- Horbett (10). moto's disease were accepted on the criteria of myxedema The Pevikon fractions were eluted in Parker's 199 with a firm smooth goiter which became smaller on thy- medium; the final volume of solution after elution was roid therapy, or in euthyroid patients with diffuse goiter 9.5 ml. This solution was dialyzed against a 0.01 M who had confirmatory evidence from 131 studies and phosphate buffer, pH 8.1, overnight. serum protein abnormalities. The diagnosis of Hashi- The column was prepared with DEAE-cellulose from moto's disease was proved histologically in 29 of the 48 Serva Entwicklungslabor, Heidelberg, and washed cases tested; of the remainder, 18 gave a positive pre- through overnight with 0.01 M phosphate buffer. The cipitin test. This series was separate from that re- dialyzed 'y-globulin solution was added to the column and ported by Pulvertaft and colleagues (2). Sera were washed on with 1 ml of buffer. A further 48 ml of obtained from 118 relatives (72 siblings, 30 offspring, buffer was passed through the column before gradient and 16 parents) of 54 propositi with thyroid disease, 46 elution was begun. The gradient was developed by run- with Hashimoto's disease, 2 with biopsy-proved focal ning a 0.10 M phosphate buffer, pH 8.1, containing 0.35 thyroiditis, 4 with thyrotoxicosis, and 2 with suspected M sodium chloride into a constant volume mixing cham- Hashimoto's disease. ber (volume, 280 ml) containing the original buffer. The Ninety-one control sera from persons without overt eluate was collected in 4-ml aliquots. The absorbance at thyroid disease were obtained from the antenatal clinic 280 mju in a 0.5 cm cell was measured in a Hilger spectro- (21 sera) and from the routine pathology laboratory. photometer. The solutions were pooled into 4 fractions Diagnoses were obtained from information in the case containing 32, 48, 40, and 130 ml, respectively. The frac- notes. Sera of 27 blood donors, 8 healthy young adult tions were concentrated approximately 20-fold overnight medical workers, 4 relatives of an 11 year old girl with by applying a vacuum outside the solutions contained in systemic lupus erythematosus, and 31 hospital patients dialysis tubing. The four concentrated fractions were were also tested. The diagnoses of the latter were: or- dialyzed against phosphate-buffered saline and then Par- thopedic conditions (6 cases), gingivitis, hepatic cir- ker's 199 medium. All operations were carried out at rhosis with aplastic anemia, dysmenorrhea, migraine, al- 4°C. The fractions and a heat-inactivated control of lergic rhinitis, ulcerative colitis, Addison's disease, paro- each were tested for cytotoxicity in the tissue culture titis, idiopathic thrombocytopenic purpura, anxiety states test. (3 cases), hypopituitarism, obesity (2 cases), lupus Sucrose gradient ultracentrifugation. Five-tenths ml of erythematosus (5 cases), diabetes mellitus, rheumatoid a 1: 5 dilution of serum was layered above 4.5 ml of a arthritis, ischemic heart disease, hypotension of unknown 12 to 36 per cent sucrose density gradient and the tubes etiology, and idiopathic hemolytic anemia. spun at 35,000 rpm in the SW 39 head of the Spinco model L ultracentrifuge for 15 hours. After centrifu- RESULTS gation the bottom of the tube was pierced, and separate 19S and 7S fractions were collected. The proteins were Requirements for complement. Heating cyto- identified by immunoelectrophoresis and tested for cytotoxic serum to 56°C for 30 minutes abolished its plasmic antibody by Coons' technique. Dialyzed frac- cytotoxic effect, and addition of fresh normal hu- tions were tested for cytotoxicity in one experiment. man or guinea pig serum to the heated serum re- Other tests for thyroid antibodies. Coons' fluorescent antibody technique was used for the determination of stored its potency. The addition of increasing cytoplasmic staining of unfixed frozen sections of toxic amounts of fresh human serum to a heated cyto- 998 FORBES, ROITT, DONIACH AND SOLOMON

TABLE I Zone electrophoresis of cytotoxic serum *

Cytotoxic factor Final dilutions Titer of cytoplasmic Fraction Analysis 6 24 120 600 3,000 stainingt 1 -y-Globulin + + 4 - - 10 2 -y-Globulin + + 1: - 50 3 y + ,2A-Globulin + 1 - - 10 4 as + #-Globulins - - - - 1 5 a, + a2-Globulins - - - - - Neg. 6 a,-Globulin + - - -- - Neg. albumin * All cells in culture killed, +; few cells surviving, A; good growth of cells,-. t Titer given as reciprocal of highest dilution of fraction giving positive staining in relation to control section treated with negative serum. toxic serum in the standard system showed that mic staining was simultaneously removed. Traces approximately 5 MHD of complement were re- of activity remained in the supernatant in a fur- quired to restore potency; thus 0.1 ml of fresh ther precipitation experiment. serum was routinely used to provide an adequate In electrophoretic separations the cytotoxic fac- excess. tor was predominantly localized to the y-globulin Serum fractionation studies. The cytotoxic fac- fractions of serum. The results of a typical sepa- tor was completely precipitated by 18 per cent ration are shown in Table I. The slow f8-globulin sodium sulfate in one experiment, while cytoplas- fraction always had low cytotoxic activity, al-

I I -2 1 3 1 4. 1 FIG. 1. CHROMATOGRAPHY ON DEAE-CELLULOSE COLUMNS OF -y-GLOBULIN FROM CYTOTOXIC SERUM OBTAINED BY ZONE ELECTROPHORESIS ON "PEVIKON" BLOCKS. Pools of eluate used in testing for cytotoxic factor in tissue culture: 1, 2, 3, 4. THYROID CYTOTOXIC AUTO-ANTIBODY 999

TABLE II TABLE IV Cytotoxicity of -y-globulin fractions obtained by Relationship of cytotoxic antibody to DEAE-cellulose column chromatography second colloid antibody

Cytotoxic factor Cytotoxic Final dilutions Second antibody colloid Fraction* 6 24 120 600 3,000 antibody Number Present Absent 1 + + 4 _ Present 39 28 11 2 - - -- _ Absent 82 11 71 3 - -- _ -

4 - - - _ - * 1, 2, 3,4 = fractions obtained by pooling eluate shown cytotoxic antibodies commonly occur together, in Figure 1. See footnote (*) to Table I. high-titer TRC or positive precipitins may be present in sera that do not contain the cytotoxic though y-globulin could not always be identified factor (Table III). This was found in two pa- in the eluate by immunoelectrophoresis. Parallel tients with Hashimoto's disease who had been tests for cytoplasmic staining showed a correla- treated with thyroxine. On the other hand, tests tion with cytotoxic activity (Table I). In four for thyroglobulin antibody were negative in 48 other experiments carried out with the same sera showing cytotoxic activity. Absorption of method the cytotoxic activity was found in the cytotoxic sera with thyroglobulin did not affect same fractions. In two of these experiments the their cytotoxic titer. Further, of 39 sera with complement-fixing and cytoplasmic antibodies were antibodies to the second colloid antigen, 28 had tested for and found to be contained in the cytotoxic activity. Conversely, of 82 sera without y-globulin fractions. colloid staining, 11 were cytotoxic (Table IV). When y-globulin fractions from two Pevikon A close relationship was found between cyto- blocks were chromatographed on DEAE-cellulose, toxicity and the presence of microsomal CF anti- (10), activity was recovered in the breakthrough body (Table V): of 86 sera giving thyroid-specific peak containing 7S y-globulin (Figure 1, Table complement fixation, all were cytotoxic except one serum with a CFT titer of ¼. One other se- Five sera were fractionated by sucrose gradient rum, from a patient with nontoxic nodular goiter centrifugation, and in each case cytoplasmic stain- having a CFT of '/'6, was not cytotoxic. This ing properties were recovered in the 7S globulins, serum also fixed complement with a saline extract while no activity could be shown in the 19S frac- of liver to a titer of ¼ 6 Cytoplasmic antibody tions. In one experiment using dialyzed frac- was absent in both of these sera. Forty-five tions in the tissue culture test, cytotoxicity was weakly cytotoxic sera gave negative results in the contained in the 7S globulins. CFT when 2 MHD of complement were used for Relationship of cytotoxic antibody to other thy- the test. However, when 13 of these sera were roid autoantibodies. Although thyroglobulin and

TABLE V TABLE III Relationship of cytotoxic factor to microsomal Relationship of cytotoxic factor to thyroglobulin complement-fixing antibody hemagglutinating antibody Cytotoxic factor factor Cytotoxic CF titer Number Present Absent Titer Number Present Absent 512 10 10 2,500,000 23 22 1 256 9 9 250,000 9 9 128 15 15 25,000 13 12 1 64 15 15 2,500 16 16 32 8 8 250 19 1 1 8 16 16 15 1 25 6 1 5 8 8 8 5 6 6 4 6 5 Negative 172 48 124 Negative 176 45 131 1000 FORBES, ROITT, DONIACH AND SOLOMON 4.Or correlation coeff.= 0248 4.Or correlation coeff. = 0.895 0 61 0 00 3.0-f 0 0 2 3.0 0 I- .- 4._ 0

x 0 x 2.01- °0 2.0 0 u-

0 0 806p 0 1.01- o4 1.0 0 c5D 0 0

0 0 1.0 2.0 3.0 1.0 2.0 3.0 log CFT titre log O cytoplasmic staining titre

FIG. 2. LEFT: CORRELATION OF CYTOTOXICITY OF HASHIMOTO SERA WITH COMPLEMENT- FIXING ABILITY. The log10 of the reciprocal of the highest serum dilution showing cytotoxicity was plotted against log,0 of the titer in the CFT test, with 11/4 minimal hemolytic doses of complement. RIGHT: CORRELATION OF CYTOTOXICITY OF HASHIMOTO SERA WITH CYTOPLASMIC STAIN- ING ABILITY. The log10 of the titer in the cytotoxic test was plotted against log,0 of the reciprocal of the highest dilution giving positive fluorescent staining of thyroid cytoplasm in Coons' sandwich technique. retested with 1¼ MHD of complement and a whereas the minimal serum dilution used in the higher antigen concentration, 9 were positive and cytotoxic test is 1: 8. 4 had become anticomplementary. In view of the Sensitivity of various types of thyroid tissue. difficulties inherent in determining the titer of cy- The results obtained with different types of thy- totoxic activity, the correlation coefficient of 0.848 roid gland are summarized in Table VII. Al- obtained between log (CFT titer) and log (cyto- though small proportions of colloid goiter and toxic titer) is good (Figure 2, left). "normal" tissues were susceptible, they were not The fluorescent test appears to be the most satisfactory for testing unknown sera, since cells sensitive and specific for microsomal antibodies, were not uniformly affected, and a variable num- and results of cytoplasmic staining agreed well ber survived in the presence of potent sera. In with cytotoxic tests (r = 0.895; Figure 2, right; three cases normal tissues were from the healthy Table VI). Of 156 sera tested by both methods, 67 were positive in both, 83 were negative by the TABLE VII two tests, while weak cytoplasmic staining was Sensitivity of various types of thyroid tissue to obtained in 6 sera in which cytotoxicity could not cytotoxic factor; analysis of 49 cultures be demonstrated. This apparent greater sensi- No. of Sensitivity* tivity of the fluorescent antibody test is undoubt- glands edly due to the fact that undiluted serum is used, Type of thyroid tissue cultured - + + + Graves' disease 25 5 20 TABLE VI Toxic nodular goiter 4 1 1 2 Nontoxic nodular goiter 11 7 4 Relationship of cytotoxic factor to Papillary. carcinoma 2 2 cytoplasmic antibody Metastasis of papillary carcinoma 1 1 Cytotoxic Hashimoto's disease 1 1 factor 4 2 2 Cytoplasmic Normal thyroid tissue antibody Number Present Absent Monkey thyroid 1 1 Present 73 67 6 * Good growth of cells in potent cytotoxic-sera, -; cells Absent 83 83 affected by potent sera only, +; cells killed by low levels of cytotoxic antibody, + +. THYROID CYTOTOXIC AUTO-ANTIBODY 1001 lobe of glands with single adenomata; two were The sensitivity of cells released by each enzyme insensitive and the other partially sensitive. Un- was compared with that of trypsin-dispersed cells. equivocally normal thyroid tissue taken during While the cells obtained by all the enzymes were surgical exploration of the neck yielded cells that sensitive to cytotoxic factor, a small percentage of were killed by the high-titer sera but not by weakly collagenase-treated cells survived in chambers active sera. Glands having a high content of mi- containing cytotoxic sera, indicating a reduced sen- crosomal antigen measured by the technique of sitivity of cells dispersed by this enzyme. The Roitt, Doniach and Couchman (15) yielded sen- best results were obtained when collagenase was sitive cells while, in general, insensitive glands mixed with trypsin. Ficin-treated cells were com- had a low antigen content. One gland with sub- pared with trypsinized cells in four experiments. stantial lymphoid infiltration yielded highly sen- Concordant results were obtained on three oc- sitive cells, although the antigen content per gram casions, but in one experiment the ficin-treated of wet tissue was in the low range. Attempts to cells were insensitive, although the trypsinized increase the sensitivity of cells from nontoxic nodu- cells from the same gland were fully susceptible to lar goiters by incubation with thyrotrophin, iodide, the cytotoxic antibody. iodine, carbimazole, and thyrotrophin plus iodide Absorption studies. Activity was absorbed in high concentration were unsuccessful. In these from a strongly cytotoxic serum (titer 1/3,000) experiments primary cultures were set up in the by mixing 0.3 ml of a 1: 10 dilution of the serum presence of potent cytotoxic sera with added com- with 0.2 ml of packed trypsinized cells from a thy- plement in the usual concentrations, and the fol- rotoxic gland. Cytotoxicity was similarly ab- lowing concentrations of these substances were sorbed out when 0.3 ml of a serum having a cy- added: thyrotrophin (Armour), 1.25 U per ml; totoxic titer of 1/600 was allowed to react at potassium iodide, 6.25 mg per ml; carbimazole, room temperature for 3 hours with 2.7 ml of a 0.25 mg per ml; Lugol's solution to give iodine 20 per cent toxic thyroid gland homogenate, the 33.3 ug per ml; Lugol's solution as above plus supernatant obtained being tested after centri- thyrotrophin, 1.25 U per ml. In each case there fugation at 105,000 G for 40 minutes. Cytotoxic was no reduction in the number of surviving cells effects were also abolished in two of three Hashi- as compared with control chambers containing moto sera when 0.2 ml of serum was absorbed with cytotoxic sera only. microsomes derived from 10 g of thyrotoxic tis- Sensitivity of autologous thyroid cells to auto- sue. Mitochondrial fractions from the same ho- antibodies. Serum containing cytotoxic factor mogenate did not absorb out the factor, nor did killed cell cultures obtained from the patient's own comparable treatment with cells or microsomal gland provided this was sensitive with known fractions from colloid goiters. positive sera. Cytotoxicity was demonstrated to Fluorescent tests on thyroid cells in culture. autologous cells in 12 experiments, and in 4 other Fluorescein-conjugated Hashimoto serum was left experiments the patient's serum having no cyto- in contact with living susceptible monolayers at toxic factor allowed good growth of autologous 370C for 45 minutes. There was no observable cells. change in the cells, and only a faint halo of fluores- The use of enzymes for cell dispersal. The pos- cence was seen when the culture was viewed by sibility that the enzymes used for dispersal might ultraviolet light. Addition of complement pro- affect the sensitivity of thyroid cells was investi- duced cytotoxic changes showing that conjugated gated; 0.25 per cent trypsin, 1 per cent papain, serum was still active. When monolayers from 0.5 per cent ficin, and 0.1 per cent collagenase in toxic and nontoxic glands were washed briefly Parker 199 medium released cells readily. Phase- and allowed to dry, the cytoplasm was weakly contrast microscopy showed that cells were rap- stained by conjugated globulins from a Hashi- idly damaged by papain unless harvested and moto serum, while no fluorescence was observed washed within 20 minutes. They were not read- with a conjugated normal serum. When conju- ily released when papain was diluted in phosphate- gated antihuman-y-globulin serum was applied af- buffered saline; with ficin, cells survived well for ter untreated Hashimoto serum (sandwich tech- 1 hour. nique), the dried cells showed bright fluorescence. 1002 FORBES, ROITT, DONIACH AND SOLOMON

Far weaker staining of the cell cytoplasm was TABLE IX seen with normal controls, although some non- Incidence of cytotoxic factor specific uptake of y-globulin occurred. No. of Cytotoxic Cultures grown in normal serum were tested sera factor daily for susceptibility to cytotoxic factor, and Condition tested present the cytoplasmic staining of dried monolayers was Hashimoto's disease 48 46 96 compared (Table VIII). Cytotoxic serum and Focal thyroiditis 6 6 complement were applied to living cells and incu- Myxedema 9 6 Thyrotoxicosis 51 34 67 bated for 30 minutes. Characteristic cytolytic Nontoxic nodular goiter 31 5 16 were seen in 24-hour old Carcinoma of thyroid 11 2 changes monolayers-i.e., Subacute thyroiditis 8 1 condensation and vesiculation of mitochondria, in- Mothers of cretins 15 1 Relatives of persons creased density of the nuclear membrane, loss of having thyroid disease 118 34 29 nuclear structure with final swelling, and rupture Lupus erythematosus without thyroid disease 4 0 of the cells. A few cells remained sensitive in 48- Lupus erythematosus hour old monolayers, but no effects were obtained with thyroid disease 3 2 in older cultures. Control series 91 8 9

TABLE VIII Comparison of sensitivity of thyrotoxic cells in culture with proven de Quervain's disease had cytotoxic fac- cytoplasmic staining of dried monolayers at 24-hour tor. Very weak cytotoxic antibody was found in intervals * 1 of 15 mothers of athyreotic cretins and the cy- was Age of culture (hours) toplasmic staining given by her serum also 24 48 72 96 weak. Eight of 91 sera (9 per cent) tested from per- Sensitivity + 1 -- overt disease had Direct Hashimoto + i: 4i sons without thyroid cytotoxic conjugate antibody. In 7 of these the antibodies were weak Direct normal - - - conjugate as judged by their inability to kill all cells in a Sandwich technique, + + + + + + primary culture. Stronger cytotoxic antibodies Hashimoto serum Sandwich technique, 4 : + not were found in a case of acute hemolytic anemia. normal serum done This serum was titered on a moderately sensitive Rabbit antiglobulin i ±i ± conjugate gland and was completely cytotoxic at % but did not affect the cells significantly at 24-. Serum * Strong cytoplasmic fluorescence, + +; weak cyto- taken after the hemolytic process had remitted un- plasmic fluorescence, +; barely perceptible fluorescence, der steroid therapy was barely cytotoxic even to 4; no fluorescence, -. cyctoplasmic highly susceptible cells. Three of these positive Fluorescent staining was more specific with di- controls were found in a group of 21 pregnant rect Hashimoto conjugates, and fluorescence was women, and they gave birth to healthy babies. maximal at 24 hours, decreasing thereafter. The The other positive controls were a 75 year old sandwich technique gave bright staining up to 48 hours and then decreased, but some staining was TABLE X Incidence of cytotoxic factor in relatives also seen with normal serum in controls. of 54 persons having thyroid disease Clinical incidence of cytotoxic factor. The in- Cytotoxic Any thyroid cidence in various conditions is presented in Table Number factor antibody* IX. Cytotoxic factor was demonstrable in all but 2 of 48 Hashimoto subjects. A high incidence was Siblings 72 19 26 34 47 also found in patients with focal thyroiditis, myx- Offspring 30 4 13 7 23 Parents 16 11 69 12 75 edema, and thyrotoxicosis. Weaker cytotoxic ac- Total 118 34 29 53 45 tivity was demonstrable in a small proportion of patients with colloid goiters and thyroid carci- * One or more tests positive for thyroid antibodies (antibodies to thyroglobulin, second colloid antigen, or cyto- noma. Only 1 of 8 patients with histologically toxic factor). THYROID CYTOTOXIC AUTO-ANTIBODY 1003

woman with cardiovascular disease, a woman with tests reflect interactions of the same antigen-anti- other thyroid antibodies whose child had systemic body system. Activity in the three tests is unaf- lupus erythematosus, and two healthy subjects. fected by prior absorption of the sera with thyro- Seven of the 8 cytotoxic control sera were tested globulin or second colloid antigen but is abolished for cytoplasmic staining and all gave positive by treatment with thyroid fractions known to have results. a high content of microsomal antigen, such as Thirty-four of 118 (29 per cent) close relatives toxic thyroid gland homogenates or microsomal of 54 persons with thyroid disease had cytotoxic preparations derived from them. Irvine (4) and antibodies. The highest incidence was found in Goudie and McCallum (5) also found that cyto- parents (69 per cent). Twelve of the parents toxic activity was removed by extracts of toxic tested were female (cytotoxic factor in 8) and 4 thyroid tissue. were male (3 cytotoxic). The incidence in sib- The variable sensitivity of thyroid glands fur- lings and offspring was 26 and 13 per cent, re- ther accords with the view that the microsomal spectively (Table X). antigen is involved both in cytotoxic activity and Cytotoxic activity was present in the sera of in cytoplasmic staining with fluorescent antibody. only 2 of 7 subjects with systemic lupus erythema- Thus the most consistently sensitive cultures are tosus and these had associated Hashimoto's derived from the glands of patients with Graves' disease. disease, although even among thyrotoxic thyroids were encountered. Cells DISCUSSION weakly sensitive glands from toxic nodular goiters were sensitive in three The antibody nature of the cytotoxic factor was of four cases, and in two cases even to weakly suggested by previous work showing that comple- cytotoxic sera. Approximately half the nontoxic ment was required for activity and that the effect nodular goiters and normal thyroid glands were was organ and species specific (1, 2). Further unaffected by Hashimoto sera, and the remainder support for this view was obtained in the present were only weakly sensitive. The degree of sen- studies by the results of serum fractionation experi- sitivity of a gland roughly parallels its content of ments. The cytotoxic factor was found to be stable microsomal CF antigen, the majority of thyro- at 560C for 30 minutes, disproving the original sug- toxic glands having a far higher content than gestion that the antibody was heat labile (1). The has normal tissue (16). The presence of an ade- factor was precipitated together with the y-globu- quate level of microsomal antigen appears to be lins by salt fractionation. After zone electropho- paramount in determining the sensitivity of the resis, virtually all of the activity was recovered in cells. Loss of susceptibility to the factor, which the y-globulins; the low cytotoxicity associated occurs in sensitive cultures after 24 to 36 hours, with the slow fl-globulin fraction may have been closely follows a marked decrease in the staining due to traces of y-globulin in concentrations inability of these cells in the fluorescent antibody sufficient for detection by immunoelectrophoresis. test. This suggests that the antigen gradually These findings are at variance with the work of disappears from the cultured cells. Leakage into Irvine (4) who failed to isolate the cytotoxic fac- the medium seems unlikely in view of the firm tor in the y-globulin fraction by both paper elec- binding of the antigen in particulate structures trophoresis and Cohn fractionation. On ultra- within the cell (17). The influence of thyroxine centrifugation the factor was localized in the 7S and of thyroid-stimulating hormone on the level fraction. Thus the cytotoxic factor has the prop- of CF antigen in the monkey thyroid (unpublished erties of a normal low-molecular weight immune results) suggests that it is related to hormonal antibody in its ability to kill the patients' own thy- activity; if the antigen is a hormone precursor, roid cells in culture. Close correlations were es- its continued metabolism might deplete the cell in tablished between cytotoxic potency, CFT titers, the absence of a precursor pool in the tissue cul- and cytoplasmic staining in individual sera. The ture environment. Denaturation of the antigen inherent difficulties of determining accurate end at the cell surface could also account for loss of points in the cytotoxic and cytoplasmic staining sensitivity, and there is some evidence that the techniques allowed for, it appears that the different cytotoxic antibody acts on cells whose surface 1004 FORBES, ROITT, DONIACH AND SOLOMON has been slightly modified by enzyme action. Pre- However, patients with focal thyroiditis have much liminary experiments, in which minute fragments lower titers of cytotoxic factor than have untreated of human thyroid were cultured by the Trowell Hashimoto subjects and it may be that high-titer raft technique in the presence of 50 per cent antibodies act synergistically with sensitized Hashimoto serum with added complement, failed lymphoid cells. The lymphocytes may injure the to show any specific tissue damage on subsequent cell membrane sufficiently [possibly during em- histological examination of the fragments. Thus peripolesis-i.e., intracellular wandering (1, 2)] enzyme treatment appears to be essential for the to allow the cytotoxic factor to enter into the cyto- preparation of sensitive cells, suggesting that sur- plasm where it exerts its full effect. It is of in- face digestion uncovers the required antigenic de- terest that Robineaux (22) was able to show by terminants. It is of interest that, in occasional ex- phase-contrast cinemicrography that slight sur- periments, collagenase or ficin-treated cells were face injury of white blood corpuscles is sufficient definitely less sensitive than were those derived to enable the lupus erythematosus factor to com- from the same gland treated with trypsin. bine with live leukocytes. Further evidence that the cell surface is the re- The non-organ specific complement-fixing an- acting site derives from the studies made with tibodies reacting with thyroid homogenates and fluorescein-conjugated Hashimoto serum. Inabil- with liver or other human and animal organs ity to penetrate the living cells prevented the se- (AICF) do not produce cytotoxic effects in cul- rum from staining the cytoplasm in healthy cultured thyroid cells, as shown by the negative re- tures, but faint surface staining was observed. In sults obtained with systemic lupus erythematosus view of the lack of penetration of antibodies into and other AICF-reacting sera. living cells, the ability of trypsinized cell suspen- Blizzard and colleagues (23) and Beierwaltes, sions to absorb out cytotoxic activity from Hashi- Dodson and Wheeler (24) put forward the sug- moto serum also argues for combination occurring gestion that congenital athyreotic cretinism may at the cell surface. Previous studies on immune be due in some cases to placental transmission of cytolysis with other cell systems such as HeLa cells cytotoxic antibodies which presumably kill the (18, 19), and immune hemolysis (20), have em- fetal thyroid during pregnancy. We therefore phasized the importance of cell surface interac- tested 15 mother and cretin-baby pairs by tissue tions with the antibody. Electron microscope culture. Only one of the mothers' sera was weakly studies using ferritin-labeled antibody to HeLa cytotoxic. Since similar weak cytotoxicity was cells (21), have shown that antibody combination demonstrated in 9 per cent of blood donors and is restricted to the cell surface until after the ad- mixed hospital patients without overt thyroid dis- dition of complement, when binding to the sub- ease, the finding in this mother is not significant. cellular elements of the cytoplasm can be ob- Further, three mothers giving birth to healthy in- served after rupture of the cell membrane. Thus fants had cytotoxic antibody during pregnancy. our tissue culture studies indicate that the same Thus the hypothesis of placental transfer of cyto- mechanisms obtain in autoimmune cytolysis as in toxic antibody as a cause of cretinism receives no heterologous immune systems. support from the present studies [see also Parker The alteration of the cell surface apparently and Beierwaltes (25)], although it is conceivable required for the demonstration of the cytotoxic that sensitized lymphocytes may occasionally over- effect makes it seem unlikely that normal thyroid come the placental barrier and destroy the fetal cells can react with cytotoxic antibodies in vivo. thyroid. Passive infusion into a monkey (14) of Hashi- SUMMARY moto serum containing high levels of cross-reacting CF antibodies indeed failed to produce thyroid in- 1. The factor in Hashimoto sera cytotoxic for jury. The fact that the cytotoxic antibodies have human thyroid cells in tissue culture has been been demonstrated in two-thirds of thyrotoxic pa- shown to be a low-molecular weight y-globulin, tients who have only a mild and nonprogressive further confirming its antibody nature. focal thyroiditis also suggests that their presence 2. The cytotoxic antibodies are identical with does not imply progressive tissue destruction. or very closely related to the antibodies that fix THYROID CYTOTOXIC AUTO-ANTIBODY 1005 complement in the presence of toxic thyroid mi- 4. Irvine, W. J. The cytotoxic factor in thyroid dis- crosomes, and are demonstrable by Coons' indi- ease. Scot. med. J. 1960, 5, 511. rect fluorescent antibody technique in unfixed 5. Goudie, R. B., and McCallum, H. M. Loss of tissue- specific autoantigens in thyroid tumors. Lancet frozen sections of toxic thyroid tissues. 1962, 1, 348. 3. Thyroid glands having a high content of mi- 6. Donnelley, M. Studies in experimental immunology crosomal antigen yielded cells sensitive to the of influenza. VII. An improved complement-fixa- cytotoxic antibody, while insensitive glands had tion technique. Aust. J. exp. Biol. med. Sci. 1951, a low antigen content. 29, 137. 4. The cytotoxic antibodies were active against 7. Mfiller-Eberhard, H. J. A new supporting medium for preparative electrophoresis. Scand. J. clin. autologous thyroid cells in culture. Lab. Invest. 1960, 12, 33. 5. The loss, with time, in sensitivity of cul- 8. Dulbecco, R., and Vogt, M. Plaque formation and tures to the cytotoxic antibodies parallels the loss isolation of pure lines with poliomyelitis viruses. in ability of the cells to stain with specific "anti- J. exp. Med. 1954, 99, 167. microsomal" sera in the Coons' fluorescent anti- 9. Scheidegger, J. J. Une micro-methode de l'immuno- body technique. electrophorese. Int. Arch. Allergy 1955, 7, 103. 6. Cytotoxic antibodies were found in 29 per 10. Fahey, J. L., and Horbett, A. P. Human gamma globulin fractionation on anion exchange cellulose cent of close relatives of patients with thyroid columns. J. biol. Chem. 1959, 234, 2645. disease. Serum from patients with systemic lupus 11. Holborow, E. J., Brown, P. C., Roitt, I. M., and erythematosus was not cytotoxic except in two in- Doniach, D. Cytoplasmic localization of "com- stances of an associated thyroiditis. plement-fixing" auto-antigen in human thyroid 7. No evidence was obtained to support the view epithelium. Brit. J. exp. Path. 1959, 40, 583. 12. Balfour, B. M., Doniach, D., Roitt, I. M., and that athyreotic cretinism results from transpla- Couchman, K. G. Fluorescent antibody studies cental transfer of these cytotoxic antibodies. in human thyroiditis: Auto-antibodies to an antigen of the thyroid colloid distinct from thyroglobulin. ACKNOWLEDG M ENTS Brit. J. exp. Path. 1961, 42, 307. 13. Fulthorpe, A. J., Roitt, I. M., Doniach, D., and We wish to thank Professor A. Kekwick and the Couchman, K. G. J. clin. Path. In press. Clinical Research Committee for facilities afforded in 14. Roitt, I. M., and Doniach, D. Human auto-immune the Institute of Clinical Research and are grateful to thyroiditis: Serological studies. Lancet 1958, 2, Professor Sir Charles Dodds and Professor F. Dickens 1027. for their unfailing support. We are greatly indebted to 15. Roitt, I. M., Doniach, D., and Couchman, K. Stud- Professor R. J. V. Pulvertaft for help and advice with ies on an intracellular thyroid auto-antigen in tissue culture techniques, and to Mr. K. G. Couchman Mechanisms of Antibody Formation. New York, and Mr. C. Shapland for skilled assistance. The work is Academic Press, 1960, p. 70. supported in part by a grant to the Middlesex Hospital Medical School from the British Empire Cancer Cam- 16. Belyavin, G., and Trotter, W. R. Investigations of thyroid antigens reacting with Hashimoto sera: Some was donated the paign. equipment generously by Evidence for an antigen other than thyroglobulin. Mary Kinross Charity Fund. Thanks are due to the Vaughan Hudson Clinical Research Trust, the Wellcome Lancet 1959, 1, 648. Trust (I.J.F.), and the Children's Hospital, Columbus, 17. Roitt, I. M., Doniach, D., Wilson, E. G., and Couch- Ohio (I.L.S.), for financial support. man, K. Biochemical studies of thyroid autoantigens. Bull. Soc. Chim. biol. (Paris) 1960, 42, 1165. REFERENCES 18. Goldberg, B., and Green, H. Immune cytolysis. I. 1. Pulvertaft, R. J. V., Doniach, D., Roitt, I. M., and The release of ribonucleoprotein particles. II. Hudson, R. V. Cytotoxic effects of Hashimoto Membrane-bounded structures arising during cell serum on human thyroid cells in tissue culture. fragmentation. J. biophys. biochem. Cytol. 1960, Lancet 1959, 2, 214. 7, 645. 2. Pulvertaft, R. J. V., Doniach, D., and Roitt, I. M. 19. Hiramoto, R., Goldstein, M. N., and Pressman, D. The cytotoxic factor in Hashimoto's disease and Limited fixation of antibody by viable cells. J. its incidence in other thyroid diseases. Brit. J. nat. Cancer Inst. 1960, 24, 255. exp. Path. 1961, 42, 496. 20. Lepow, I. H., and Ross, A. Studies on immune cel- 3. Irvine, W. J. An investigation of the pathogenesis lular injury. II. Functional role of C'1 esterase of Hashimoto's disease by thyroid tissue culture. in immune cytotoxicity. J. exp. Med. 1960, 112, J. Endocr. 1960, 20, 83. 1107. 1006 FORBES, ROITT, DONIACH AND SOLOMON 21. Easton, J., Goldberg, B., and Green, H. Electron 23. Blizzard, R. M., Chandler, R. W., Landing, B. H., microscopic localization of ferritin-labeled anti- Pettit, M.D., and West, C. D. Maternal autoim- body in ascites tumor cells. Fed. Proc. 1961, 20, munization to thyroid as a probable cause of 16. athyrotic cretinism. New Engl. J. Med. 1960, 263, 327. 22. Robineaux, R. Study of L. E. cell formation by phase 24. Beierwaltes, W. H., Dodson, V. N., and Wheeler, contrast microcinematography in Henry Ford Hos- A. H. Thyroid autoantibodies in the families of pital International Symposium on Mechanisms of cretins. J. clin. Endocr. 1959, 19, 179. Hypersensitivity, J. H. Shaffer, G. A. LoGrippo, 25. Parker, R. H., and Beierwaltes, W. H. Thyroid and M. W. Chase, Eds. Boston, Little, Brown, antibodies during pregnancy and in the newborn. 1959, p. 371. J. clin. Endocr. 1961, 21, 792.