Cliii. exp. Immunol. (1978) 32, 159-168.
Receptors for fluoresceinated human thyroglobulin in peripheral blood lymphocytes G. B. SALABE, H.
SALABfE,
L. ACCINNI & R. DOMINICI Centro di Fisiopatologia Tiroidea CNR, Clinica Medica II, Universitd di Roma, Rome, Italy
(Received 15 May 1977)
SUMMARY
Fluoresceinated human native and desialylated thyroglobulin were incubated with peripheral blood lymphocytes. 1 % of the lymphocytes, in twenty samples from normal human blood donors, showed a bright granular fluorescence where neither the number nor pattern of fluorescence differed from lymphocytes from the blood of thyroiditis patients. Fluoresceinated albumin and y-globulin did not bind to the lymphocytes, and a 500-fold excess of native non-fluoresceinated thyroglobulin inhibited the binding and pre-incubation with anti-IgM serum abolished it. Binding with desialylated thyroglobulin was negligible, and the pattern of fluorescence was pale and uniform. Analysis by sucrose gradient centrifugation and double diffusion in agar gel showed that fluorescein dissociates thyroglobulin into 12S fragments and reduces its immunoreaction with autoantibodies. It can therefore be concluded that the 12S molecule produced by fluoresceination maintains its determinants for lymphocyte receptors, whereas further dissociation, as in desialylated fluoresceinated thyroglobulin, leads to a marked reduction in the binding with lymphocytes. INTRODUCTION Antigen-binding cells identified as some of the circulating lymphocytes have been considered as an expression of the immunological status. In fact, immunization in several animal species increases the number of antigen-binding cells, and the depletion of specific antigen-binding lymphocytes abolishes the immune response towards specific antigens. Tolerant antigens, on the other hand, have a negligible number of binding lymphocytes (Ada & Cooper, 1971; Warner, 1974). The binding of thyroglobulin to peripheral (Bankhurst, Torrigiani & Allison, 1973; Roberts, Whitting & Mackay, 1973), spleen (Clagett & Weigle, 1974) and thymus (Roberts et al., 1973) lymphocytes has been revealed by the autoradiographic technique in the mouse and human (Bankhurst et al., 1973; Urbaniak, Penhale & Irvine, 1973) with highly labelled (10 pCi/,ug) 125I-labelled thyroglobulin. Binding cells were identified as B lymphocytes (Bankhurst et al., 1973; Clagett & Weigle, 1974). In mice, immunization with autologous or heterologous thyroglobulin produces an increase of thyroglobulin-binding lymphocytes (Clagett & Weigle, 1974). In patients with chronic autoimmune thyroiditis, thyroglobulin-binding lymphocytes in the peripheral blood were seen to be either increased (Roberts et al., 1973), or unchanged with respect to normal subjects (Urbaniak et al., 1973). Following immunization with homologous thyroglobulin, the strains of rats with a high susceptibility to thyroiditis do not show a larger number of thyroglobulin-binding cells than in non-susceptible strains (Penhale etal., 1975). Correspondence: Dr G. B. Salabe, Centro di Fisiopatologia Tiroidea CNR, Clinica Medica II, Universita di Roma, Rome, Italy. 0099-9140/78/0400-0159$02.00 @) 1978 Blackwell Scientific Publications
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These investigations on thyroglobulin-binding lymphocytes have been carried out so far by autoradiography with 125I-labelled thyroglobulin. No report has yet appeared on the study of thyroglobulin binding to peripheral lymphocytes using fluorescence techniques. In the present paper, the number, specificity, nature of lymphocyte receptors and the binding to fluoresceinated thyroglobulin were studied in normal and thyroiditis patients. Furthermore, chemical and immunochemical properties of the fluoresceinated binding antigen, using sucrose gradient analysis and double diffusion in agar gel, which have not been reported in studies carried out with iodinated antigen, will be described. The binding of desialylated thyroglobulin to peripheral lymphocytes has been investigated, in order to find the receptors for antigenic determinants masked by sialic acid on the lymphocytes; as was previously found in the serum antibodies from thyroiditis patients (Salabe et aL, 1976b). MATERIALS AND METHODS Fluorescein isothiocyanate (FITC) was obtained from BDH (England); the human serum albumin, twice crystallized, from Sigma and the human gamma globulin from Behringwerke AG (Marburg-Lahn, West Germany). The Ficoll-Urovison solution was prepared by dissolving 9-239 g of Ficoll (Pharmacia, Uppsala) in 119-4 ml of distilled water and 25 ml of 58% solution of Urovison (Schering SPA). The density of the solution was 1-077 g/ml at room temperature. Hanks' solution was obtained from Difco (U.S.A.) and the thyroglobulin haemagglutination test kits from Wellcome (Beckenham, England). Rabbit anti-human thyroglobulin serum was prepared as previously described (Salabe et al., 1976b) and absorbed with 0 1 ml of normal human serum. A precipitating autoantiserum was obtained from a patient with chronic thyroiditis. Highly specific anti-human y and [± chain from rabbits were supplied by Behringwerke AG (Marburg-Lahn, West Germany). Liquemin Roche was used (20 [LI per 10 ml or 10 u/ml of whole blood). Fluoresceination of thyroglobulin. A single human thyroglobulin extracted from a follicular adenoma which did not concentrate iodine at the scintiscan was used throughout the experiments. Bovine thyroglobulin was prepared from a pool of beef thyroid gland harvested from adult animals from the local slaughterhouse. Thyroglobulin from a crude saline extract (2 ml/g of minced tissue) was purified by salting-out (1.4-1-8 M S04(NH4)2) followed by chromatography on Sephadex G-200 (Salabe et al. 1976b). Analysis of the purity of the preparation by analytical ultracentrifuigation (Beckmann Spinco model E) showed that 90% of the thyroglobulin had a sedimentation rate of 19S and 10% of 4-7S. Polyacrylamide gel electrophoresis (performed in a 4% gel, pH 8-9, for 90 min at room temperature with 2-5 mA per tube) showed a single band with an electrophoretic mobility identical to that of a marker thyroglobulin. Native thyroglobulin was digested with Vibrio cholera neuraminidase according to the technique previously described (Tarutani & Shulman, 1971). The sialic acid content by the thiobarbituric assay after neuraminidase digestion was undetectable (Warren, 1959). Native and desialylated thyroglobulin, human albumin and gamma globulin were fluoresceinated by incubating a 05-1% solution of the proteins in 0 5 M NaCl, with a 0-1 M bicarbonate buffer, pH 8-6, with varying amounts (20-50 ±g/mg of thyroglobulin) of fluorescein isothiocyanate (FITC), added in powder form, for 16 hr at 4°C under gentle stirring (Coons & Kaplan, 1950; Holborow & Johnson, 1967). After incubation, the solution was extensively dialysed against phosphate buffer 0-02 M and KC 0 1 M, pH 7-4, until the diffusate no longer adsorbed at 492 nm. The concentration offluorescein in the solution ([Lg/ml) was calculated using the formula: A492 -(A320/2). fluorescein (j±g/ml) = 0-20 The concentration of protein in the solution was measured with the method of Lowry et al. (1951) or by the biuret technique (Gornal, Bardowill & David, 1949), using human albumin in the standard curve. The content of fluorescein in the protein varied from 5-15 [Lg/mg. Iodination of thyroglobulin. The iodination of the thyroglobulin was carried out with lactoperoxidase and glucose-glucose oxidase as the H202-generating system, as previously described (Pommier, Deme & Nunez, 1973; Salabe et al., 1976b). The incubation mixture contained 2 x 10-9 mol of KI, 1 mCi of 125I, carrier-free, 10-9 mol of thyroglobulin, 750 ,ug of glucose, 2 ,ug of lactoperoxidase and 2 ,ug of glucose oxidase per ml of solution. The specific activity of the labelled thyroglobulin was 2-5 mCi/mg; 0 5-1 atom of iodine per molecule of thyroglobulin. The labelled thyroglobulin was analysed by sucrose gradient centrifugation, in order to evaluate the extent of denaturation. Preparation of lymphocytes. Lymphocytes were prepared according to the method of Boyum (1968), with minor modifications. Human lymphocytes were from young blood donors at the transfusion unit or from thyroiditis patients seen at the out-patient department. The diagnosis of thyroiditis was established on the basis of a firm lobulated goitre and a high titre of anti-thyroglobulin antibodies (tanned red cell haemagglutination with a serum dilution of 1: 25 x 106). In one case, diagnosis was also confirmed by biopsy. Horse or bovine lymphocytes were prepared from blood harvested from the local slaughterhouse. 6 ml of heparinized blood were layered over 2-5 ml of Ficoll-Urovison (p = 1-077 kg/m3) in a 10 ml conical glass tube and centrifuged (Martin Christ IKS) at 1000 g at 4°C for 30 min. Before layering human blood was diluted 1: 3 with Hanks'
Receptorsforfluoresceinated human thyroglobulin
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solution, horse blood was diluted 1: 1 and beef blood was used undiluted. At the interfaces between the Hanks' solution and Ficoll-Urovison, an opalescent ring of lymphocytes was formed, whereas red blood cells and macrophages sedimented to the bottom of the tube. The plasma and Hanks' solution were removed, the opalescent ring was collected and washed twice with Hanks' solution (7-8 ml) and once with phosphate-buffered saline (PBS) (KH2PO4-Na2HPO4, 015 M, pH 7 4) in the presence of sodium azide, 0-1%. After each wash, the solution was centrifuged at 150 g at 40 for 15 min. After the final wash the lymphocytes were suspended in PBS,with 0-1% sodium azide at a concentration of 107 per ml. The yield of lymphocytes was calculated in a Thomas-Zeiss chamber. The cells, stained by 0-01% methyl violet in citric acid, 0 1 M, were counted in both the whole blood and in the lymphocyte preparation; the yield was 15-20 x 106 lymphocytes per 10 ml of blood. The purity of the lymphocytes was evaluated in the preparation smeared by the cytocentrifuge (cytocentrifuge, Shandon) and stained with Giesma-May-Grunwald; macrophage contamination was less than 1%. The viability test was performed by incubating 1: 1 volumes of trypan blue and the lymphocytes suspension for 10 min at room temperature. Lymphocyte incubation. From 20 to 200 ±g of fluoresceinated proteins were incubated in 0-2 ml of PBS with 2 x 106 lymphocytes at 0C and in the presence of 0- 1% sodium azide. At the end of the incubation period (30 min), the lymphocytes were washed three times in 0-2 ml of PBS with sodium azide, to remove the excess of fluoresceinated unbound proteins. After each wash, the lymphocytes were run for 10 min at 150g. The inhibition of binding was performed by pre-incubation of the lymphocytes for 30 min at 4VC with rabbit anti-human IgM (0.5 ml per 5 x 106 lymphocytes) or an excess of cold, native and desialylated thyroglobulin (5-50 mg). Following the addition of the fluoresceinated protein, the mixture was incubated for an additional 30 min and washed as previously described. After the final wash, the pellet was suspended in 0-2 ml PBS with 0-1 % sodium azide. In the experiments aimed at observing the capping, the lymphocytes were incubated without sodium azide at 370C for periods offrom 10 min up to 12 hr. The lymphocytes were observed and counted under covered slides, both by fluorescence with incident light and by light microscopy, and the percentage of fluorescent cells evaluated. 2000 cells were counted for each sample with a cell counter (Clay-Adams); monocytes and cell clumps were avoided. Sucrose gradient ultracentrifugation and immunochemical properties of native and desialylated thyroglobulin. The double diffusion in agar gel was performed in 1% agar 0 04 M phosphate buffer, pH 7-1. Agar was added with 3% polyethylene glycol. Precipitin lines were stained with bromophenol blue. The sedimentation properties were analysed by linear sucrose gradient centrifugation (Spinco model L2, rotor SW27), with gradients of 5-28% or 10-40% at 26,000 rev/min at 220C for 12 hr, or 20C for 24 hr. Thirty 1 ml fractions were collected. Each fraction was estimated at 280 and 492 nm, and the "251-labelled thyroglobulin, run as the internal marker, was evaluated in a well-type scintillation counter.
RESULTS Physical and immunochemicalproperties offluoresceinated thyroglobulin The sucrose gradient centrifugation profile (Fig. 1) showed that upon fluoresceination native thyroglobulin changes its sedimentation properties: most of the fluorescence in the gradient is found in the 12S region, with only 10% remaining in the 19S region and the rest migrating with a sedimentation lower than 12S. The preparation shown in Fig. 1 had a ratio of fluorescein: protein of 15 pg/mg. At a lower level of fluoresceination (ratio fluorescein: protein of 6 jig/mg), the 19S sedimentation properties of thyroglobulin were maintained. Bovine thyroglobulin was fluoresceinated with a molar ratio of 6 and behaved as a 19S fragment in the sucrose gradient. The sucrose gradient of thyroglobulin fluoresceinated after desialylation with a ratio of 15 jig/mg revealed that the sedimentation properties of this protein were remarkably affected, and most of the absorbancy at 492 nm could not be identified in a peak and remained at the top of the gradient. Experiments were also carried out by double diffusion in agar gel to investigate the immunochemical properties of the fluoresceinated thyroglobulin (ratio of fluorescein: protein of 15 jg/mg). With rabbit anti-native human thyroglobulin antiserum (Fig. 2a), native and fluoresceinated thyroglobulin reacted identically, whereas only partial identity was seen with desialylated fluoresceinated thyroglobulin: precipitin line of native spurred over desialylated fluoresceinated thyroglobulin. With a human antithyroglobulin serum (Fig. 2b), only a faint line with fluoresceinated thyroglobulin was shown, and it did not precipitate in agar with fluoresceinated desialylated thyroglobulin. Bovine native and fluoresceinated thyroglobulin (ratioof fluorescein: protein of 6 jg/mg) reacts identically with rabbit anti-native bovine thyroglobulin. T.
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05
0*4
Cj 0~) ~0 .11
03 C 5x
.E
02
11-
a)
0-
15 10 Tube number
FIG. 1. Sucrose gradient ultracentrifugation pattern of fluoresceinated native and desialylated human thyroglobulin (SW27 rotor, gradient 10-40%, 26,000 rev/min at 220C for 12 hr). Samples were run in separate tubes of the same rotor, fractions of 1 ml were collected. Sedimentation rates were estimated by an internal 1251 marker formed by 19S and 12S human thyroglobulin labelled with lactoperoxidase. (A) 19S thyroglobulin: radioactivity vs tube number. (0) Absorbancy of desialylated thyroglobulin vs tube number; (0) absorbancy ofnative thyroglobulin vs tube number.
FIG. 2. Double diffusion in agar gel (0-04 m phospate, pH 7-4) between anti-thyroglobulin antisera and native and fluoresceinated thyroglobulins. (a) Rabbit antiserum to human 19S thyroglobulin (R) and (b) human thyroiditis serum (H) was tested with 0-1% solution of native (1), native fluoresceinated (2) and desialylated fluoresceinated thyroglobulin (3).
Number, pattern and species specificity of the binding offluoresceinated thyroglobulins to lymphocytes The number of lymphocytes binding thyroglobulin appeared to vary considerably. In twelve samples from normal blood with the same thyroglobulin preparation, fluoresceinated with 15 jig of fluorescein per mg of thyroglobulin and incubated at a concentration of 04 mg of thyroglobulin per 101 lymphocytes, the number ranged between 0-5 and 2% lymphocytes, the average being 0-99±0O4. In four thyroiditis samples examined under the same conditions, the values ranged between O'5 and 1~4%, with an average
Receptorsforfluoresceinated human thyroglobulin
163
FIG. 3. Peripheral blood lymphocytes from normal subjects incubated with fluoresceinated thyroglobulin. Two types of staining are visible at the surface of the lymphocyte: (a) granular and (b) rim. (Magnification x 800.)
of 0 9404. The number of thyroglobulin-binding lymphocytes increased with an increase in the thyroglobulin concentration (Table 1) and approached a plateau at 2 mg/ml. Below 0 05 mg/ml, the fluorescent lymphocytes were difficult to evaluate. The number of thyroglobulin-binding lymphocytes decreased and the fluorescence became pale when the cells were incubated with thyroglobulin labelled with 6 jg of fluorescein per mg of protein, instead of 15 g/ml. The most common pattern of fluorescence observed was the granular type, in which bright spots of various size and number were seen on a pale background (Fig. 3a). More rarely, the fluorescence was more diffuse on the cell surface, with a rim pattern (Fig. 3b). Finally, in some preparations, patches of fluorescence were observed on the cell. Incubation at 370C without sodium azide for 30 min to 12 hr did not produce capping of the fluorescence: there was, instead, a decrease in the number of granules on the fluorescent cells. Increasing the concentration of fluoresceinated antigens from 0 1 to 2 mg/ml for 107 lymphocytes did not change the pattern of fluorescence. Incubation of lymphocytes with desialylated thyroglobulin showed that this protein binds fewer lymphocytes than the control, and the fluorescence became pale and uniform (Table 1). TABLE 1. Effect of antigen concentration on the number of thyro-
globulin-binding lymphocytes
Fluoresceinated thyroglobulin
Native thyroglobulin-binding lymphocytes
Desialylated thyroglobulin-binding lymphocytes
(mg/ml)
(%)
(%)
0*1
13 3-3 50
0-6
05 2-0
0*7 0-6
The same preparation of human lymphocytes was subdivided into six aliquots of 2 x 106 lymphocytes, each being incubated in 0-2 ml of PBS plus 0-1% sodium azide with 0 1-2 mg of fluoresceinated thyroglobulin (15 [Lg of fluorescein per mg of thyroglobulin). After 30 min, the cells were washed and observed under a covered slide under the fluorescence microscope.
G. B. Salabel et ad.
164
TABLE 2. Binding of human lymphocytes to different fluoresceinated proteins
Fluorescein Protein (and amount in mg/ml)
Human thyroglobulin (0 1) Bovine thyroglobulin (1 0) Human albumin (2-3) Human y-globulin (0-15) Human thyroglobulin (native) (50) Human thyroglobulin plus anti-IgM (0 5 ml) (0 1)
(Gg/ml)
Thyroglobulin-binding lymphocytes (%)
15 39 11-4 16 0 15
2-8 0-8 0-01 0-0-1 0-6 0
Fluoresceinated antigens were incubated for 30 min at 00C in 0-2 ml of PBS containing 0-1% sodium azide and 2-5 x 106 lymphocyte. After incubation, the cells were washed and observed under a covered slide under the fluorescent microscope. The amounts of fluorescein incorporated in each protein was variable, therefore higher concentrations of bovine thyroglobulin and human albumin, which had a lower content of fluorescein, were added to the incubation mixture. In the inhibition experiments, native human thyroglobulin was added before fluoresceinated thyroglobulin.
Experiments designed to establish the specificity of the interaction between lymphocytes and fluoresceinated human thyroglobulin are shown in Table 2. Bovine thyroglobulin bound to a lesser extent than human thyroglobulin, whereas human albumin and gamma globulin showed negligible binding. Inhibition of binding occurred only with a 500-fold excess of native thyroglobulin. The amount of each protein added was varied, in order to keep the fluorescein concentration in the incubation mixture constant. Pre-incubation of lymphocytes with rabbit anti-human IgM serum abolished the binding with fluoresceinated thyroglobulin in both normal and thyroiditis serum. The same preparation of human thyroglobulin (15 jig of fluorescein per mg) employed in the previous experiments was used to compare the binding of human lymphocytes with horse and bovine lymphocytes (Table 3). As expected, the number of thyroglobulin-binding lymphocytes increased as the thyroglobulin concentration in the incubation medium was increased. The number of thyroglobulin-binding lymphocytes was higher with human than with horse or bovine lymphocytes. In contrast, bovine thyroglobulin (6 jig of fluorescein per mg), which is 19S, bound only to heterologous (horse and human) but not to homologous lymphocytes. TABLE 3. Species specificity of lymphocytes binding thyroglo-
bulin Lymphocytes (%)
Thyroglobulin (mg/ml) Human 0.1
0-3-0-5 10 Bovine 0.3 1-15
Human
Horse
Bovine
1-3
0-8 1-2 2-6
04 0-7
3.3 5.0
-
n.d. n.d. 0-8 Lymphocytes (2 x 106) were incubated in PBS in 0-2 ml for 30 min at 0°C in 0-1% sodium azide. Human thyroglobulin was mostly dissociated as 12S (50%) whereas bovine kept its sedimentation rate of 19S. Fluorescein: protein ratio (,ug/mg) was 15 for the human and 6 for the bovine thyroglobulin. n.d. = Not detectable, (-) not done. -
0-8
165
Receptors forfluoresceinated human thyroglobulin TABLE 4. Numbers and conditions of incubation of the thyroglobulin-binding lymphocytes
Thyroglobulin* (tag/ml) ([±Ci/jg)
Lymphocytes (x 106 per ml)
0-2-025
75-91
10
0-2-0-25
28-116
10
1-2 (microsomes) 1-2 0-5-1
1
10
1 10-15
10 10
100
6-11
50
100 0-5-2-5
6-11 33-85
50 100
0 5-2 5
33-85
100
10(§
10
Source of
lymphocytes Total peripheral blood (rat) Total peripheral blood
Thyroglobulin binding lymphocytes (%) Normal Thyroiditic -
0-08 (human) 0035 Total peripheral blood (human) Thyroid (human) 0-02 Total peripheral blood (human) 0 34 Total peripheral blood (human) 20-0-5: Thymus (human) Thymus (mouse) < 0.001 (mouse), 0 03 (bovine) 0-068 (mouse), Spleen (mouse) 0-038 (bovine), 0-13 (human) 09 Total peripheral blood (human)
References
0-2-05
Penhale et al. (1975)
0-08
Urbaniak et al. (1973)
0 11
Khalid et al. (1976)
n.d.t
Khalid et al. (1976) Bankhurst et al. (1973)
2-3
Roberts et al. (1973) Roberts et al. (1973) Clagett & Weigle (1974)
0 34
Clagett & Weigle (1974)
09
Salabe et al. (1976a)
* Unless otherwise specified, the lymphocytes were incubated with homologous thyroglobulin. t Not detectable. t At birth and in the adult respectively. § Flourescein labelled (15 Lg/mg)
Better inhibition of the fluorescence was obtained with desialylated thyroglobulin. In one experiment, pre-incubation of the lymphocytes with 4 mg of native thyroglobulin was ineffective in reducing the number of fluoresceinated lymphocytes (1.5%), whereas after pre-incubation with desialylated thyroglobulin the binding of fluoresceinated native thyroglobulin with the lymphocytes was completely abolished. Sucrose gradient centrifugation of this particular preparation showed that desialylated thyroglobulin was dissociated and composed of 20% 19S and 50% 12S, a pattern similar to that of native fluoresceinated thyroglobulin.
DISCUSSION The present observations confirm the findings of Bankhurst et al. (1973) and Clagett & Weigle (1974) on the existence of a specific thyroglobulin receptor on lymphocytes. However, the number of thyroglobulin-binding lymphocytes in our experiments is ten to fifty times higher. This difference could be due to the different techniques employed (Table 4). For instance, the autoradiographic technique used by Bankhurst et al. (1973) and Clagett & Weigle (1974) gave lower estimates since the concentration of antigen in the incubation medium was 40-200 times lower than in our experiments, thus selecting only the more avid receptor. Alternatively, the receptor determinant of thyroglobulin may be damaged during 1251 iodination. When the same thyroglobulin concentrations were employed (0.10 mg/ml), a comparable number of thyroglobulin-binding lymphocytes were found (Roberts et al., 1973). The number of thyroglobulin-binding lymphocytes was highly variable. Taking into account that a standardized procedure was used in the experiments and that the cells were always counted by the same observer, and, furthermore, that the same preparation of lymphocytes repeatedly counted gave
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G. B. Salabe et al.
reproducible results, it is thus possible that the number of thyroglobulin receptor-carrying lymphocytes has a considerable individual variability. Cytophilic antibodies did not seem to increase the number of positive lymphocytes, since the average number of thyroglobulin-binding lymphocytes did not differ between normal and thyroiditis subjects in which there was a very high titre of anti-thyroglobulin antibodies. All normal subjects examined in our study had negative anti-thyroglobulin antibodies according to the TRC test; however, small quantities of circulating anti-thyroglobulin antibodies may not be detectable by TRC, therefore the possibility still existed that the number of thyroglobulin-binding lymphocytes in our experiments was increased by cytophylic antibodies. In unpublished experiments performed in our laboratory, we were unable to demonstrate any fluorescent lymphocytes after incubation of the peripheral normal lymphocytes with fluoresceinated human gamma globulin purified from serum with high titres of anti-thyroglobulin antibody. This finding limits the hypothesis that cytophylic antibodies play a role in the binding of thyroglobulin to the peripheral lymphocytes. Antigen-binding cells amongst circulating lymphocytes have been identified as an expression of the immunological status, which was demonstrated by the fact that immunization in several animal species increased the number of antigen-binding cells (Davie, Rosenthal & Paul, 1971) and the depletion of specific antigen-binding lymphocytes abolishes the immune response towards specific antigens (Ada & Byrt, 1969). With heterologous thyroglobulin an increase in antigen-binding cells was observed in mice after immunization (Clagett & Weigle, 1974). However, in rats immunized with homologous thyroglobulin in CFA, thyroiditis-susceptible strains exhibited the same number of antigen-binding lymphocytes as the non-susceptible strains (Penhale et al., 1975). Furthermore, in man, it was not possible in the present or previous studies (Urbaniak et al., 1973) to demonstrate an increase in the thyroglobulin-binding lymphocytes in the blood of patients with chronic autoimmune thyroiditis. Even lymphocytes extracted from a thyroiditic thyroid gland showed a decreased binding to thyroid microsomal antigen (Khalid, Hamilton & Cauchin, 1976). Roberts et al. (1973), on the other hand, found an increase in the thyroglobulin-binding lymphocytes. It might be suggested that in thyroiditis patients the excess of thyroglobulin or other thyroid antigens, circulating or in the thyroid, might block the receptors. An increase in circulating thyroglobulin has been demonstrated in thyroid autoimmune diseases, such as thyroiditis & Graves' disease (Van Herle, Uller & Matthews, 1973). A marked decrease in the number of binding lymphocytes has been observed with desialylated thyroglobulin. The effect of desialylation on the number of thyroglobulin-binding lymphocytes might be due to the sialic acid per se, or to the denaturation of determinants for receptors produced by fluoresceination on desialylated thyroglobulin. This latter interpretation seems to be the more likely, as the immunochemical properties of fluoresceinated desialylated thyroglobulin were altered in immunodiffusion, and previous studies have shown that the removal of sialic acid does not change the immunoreaction with circulating autoantibodies, but in some cases it enhances it, suggesting that sialic acid masks the antigenic determinants (Salabe et al. 1976b). In the present study, attention was focused on the physical properties of thyroglobulin binding to the lymphocytes. Analysis of the fluoresceinated thyroglobulin revealed that a large proportion of this antigen dissociated as a 12S fragment. It seems therefore that the 12S subunits maintains the determinants for the lymphocyte receptors, although some of the determinants for antibodies were damaged, as demonstrated by the lack of immunoprecipitation with autoantibodies in double diffusion in agar gel. Specific determinants for lymphocyte receptors on the 12S subunits are supported by indirect evidence, i.e. bovine lymphocytes do not bind to bovine thyroglobulin, which by sucrose gradient centrifugation was showed to be 19S. Non-fluoresceinated 19S thyroglobulin inhibits fluorescence at a very high concentration, whereas desialylated thyroglobulin-which is mostly dissociated as 12S fragment-inhibits the fluorescence at low concentrations. These preliminary observations might lead to further experiments aimed at establishing specific determinants on the thyroglobulin molecule. Experiments since carried out in this laboratory with purified isolated 19S and 12S thyroglobulin labelled with 1251 showed that both molecules bind with lymphocytes to the same extent, and therefore it can be concluded that
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receptors on the lymphocytes bind to determinants exposed on native 19S or dissociated in 12S thyroglobulin. Denaturation, as occurs with desialylated thyroglobulin, abolishes the reaction of receptors for thyroglobulin. According to the current view (Allison, 1973; Weigle, 1973) on the production of autoimmunity at the lymphocyte receptor level, these antigens which, like thyroglobulins, circulate at low concentration in normal subjects carry determinants specific for receptors on the B cells but not on the T cells, which are made unresponsive by the low concentration of circulating antigen. T lymphocytes become responsive when a heterologous determinant emerges from a normal constituent and reacts with specific receptors on the T cells, which in turn help or derepress the B cells and stimulate them to produce antibodies towards the normal constituent. Recent findings support this hypothesis: nude mice were unable to respond to a pool of heterologous thyroglobulin, although they possess B cells with specific receptors for mice thyroglobulin (Clagett & Weigle, 1974). However, other experiments indicate another pathway in the production of autoantibodies (de Heer & Edgington, 1976). The red blood cells of mice possess native and cryptic antigens. In the normal, animal specific receptors on B and T cells are found for the cryptic antigen whereas the receptors for native antigen are only on the B but not on the T cells. Receptors on the T cells are found only in the NZB strain, which develops haemolytic autoimmune anaemia. Furthermore, it has been found that only lymphocytes from the NZB strain produce in vitro antibodies to the native antigen (de Heer & Edgington, 1976). From these studies it emerges that autoimmunity results when a particular population of T cells is present with receptors for the native antigen. Future experiments with highly purified native thyroglobulin tested with isolated B and T cells will show whether T receptors for native thyroglobulin are peculiar only to thyroiditis patients. In our experiments, it was demonstrated that the thyroglobulin receptor binds to the native antigen. Future experiments with B and T cells purified from the blood of thyroiditis patients and native thyroid antigens will contribute to clarify which of the two proposed mechanisms is operating in thyroiditis: the appearance of a heterologous determinant on a thyroid antigen or a T-cell subpopulation with receptors for the native thyroid antigen. However, the results of preliminary experiments (Wick & Richter, 1976) have recently shown that in the spleen of obese chickens with spontaneous autoimmune thyroiditis only B lymphocytes with receptors for thyroglobulin are present. We would like to thank Mr L. Corvo for skilful technical assistance.
REFERENCES ADA, G.L. & BYRT, P. (1969) Specific inactivation of antigen reactive cells with 125I-labelled antigen. Nature (Lond.), 222, 1291. ADA, G.L. & COOPER, M.G. (1971) Antigen binding cells in tolerance and immunity. Ann. N. Y. Acad. Sci. 181,96. ALLISON, A.C. (1973) Mechanism of tolerance and autoimmunity Ann. rheum. Dis. 32, 283. BANKHURST, A.D., TORRIGIANI, G. & ALLISON, A.C. (1973) Lymphocytes binding human thyroglobulin in healthy people and its relevance to tolerance for autoantibodies. Lancet, i, 226. BoYuM, A. (1968) Separation of lymphocytes from blood and bone marrow. Scand.]. clin. Lab. Invest. 21, Suppl. 97, 1. CLAGETT, J.A. & WEIGLE, W.O. (1974) Roles of T and B lymphocytes in the termination of unresponsiveness to autologous thyroglobulin in mice.j. exp. Med. 139, 643. COONS, A.H. & KAPLAN, M.H. (1950) Localization of antigen in tissue cells. II. Improvements in a method for the detection of antigen by means of fluorescent antibody. J. exp. Med. 91, 1. DAVIE, J.M.A., ROSENTHAL, S. & PAUL, W.E. (1971) Receptors on immunocompetent cells: specificity and
nature of receptors on dinitrophenylated guinea-pig albumin. '25I-binding cells of immunized guinea-pigs. 3. exp. Med. 134,517. GORNAL, A.J., BARDOWILL, C.J. & DAVID, M.M. (1949) Determination of serum proteins by means of the biuret reaction. J7. biol. Chem. 177, 751. HEER, D.H. DE & EDGINGTON, T.S. (1976) Specific antigenbinding and antibody-secreting lymphocytes associated with the erythrocyte autoantibody responses of NZB and genetically unrelated mice. J7. Immunol. 116, 1051. HOLBOROW, E.J. & JOHNSON, G.D. (1967) Immunofluorescence. Handbook of experimental Immunology (ed. D.M. Weir). p. 571. Blackwell Scientific Publications Ltd, Oxford. KHALID, B.A.K., HAMILTON, N.T. & CAUCHI, M.N. (1976) Binding of thyroid microsomes by lymphocytes from patients with thyroid disease and normal subjects. Clin. exp. Immunol. 23, 28. LowRy, O.H., ROSENBROUGH, N.J., FARR, A.L. & RANDALL, R.J. (1951) Protein measurements with a Folin phenol reagent. 7. biol. Chem. 193, 265. PENHALE, W.J., FARMER, A., URBANIAK, S.J. & IRVINE, W.J.
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(1975) Susceptibility of inbred rat strains to experimental thyroiditis; quantitation of thyroglobulin-binding cells and assessement of T-cell function in susceptible and non-susceptible strains. Clin. exp. Immunol. 19, 179. POMMIER, J., DEME, D. & NUNEz, J. (1973) Effect of iodine concentration on thyroxine synthesis catalyzed by thyroid peroxidase. Europ.j. Biochem. 37,406. ROBERTS, I.M., WHITTING, S. & MACKAY, I.B. (1973) Tolerance to an autoantigen: thyroglobulin antigen binding lymphocytes in thymus and blood in health and autoimmune disease. Lancet, ii, 936. SALABE, G.B. (1975) Immunochemistry of the interaction of thyroglobulin (Tg) and its auto-and hetero antibodies. Acta Endocr. 79, Suppl., 196. SALABE, H., AccINmI, L., DOMINICI, R. & SALABE', G.B. (1976a) Thyroglobulin binding lymphocytes in normal individuals and thyroiditis patients. Acta Endocr. (Copenh.), 82, Suppl., 204. SALABE, H., DOMINICI, R. & SALABE, G.B. (1976b) Immunological properties of Tg carbohydrates: enhancement of Tg immunoreaction by removal of sialic acid. Clin. exp. Immunol. 25,234. TARUTANI, 0. & SHULMAN, S. (1971) Properties of carbo-
hydrate-stripped thyroglobulin. I. Preparation and physicochemical properties of desialized thyroglobulin. Biochem. biophys. Acta (Amst.), 229, 642. URBANIAK, S.J., PENHALE, W.J. & IRVINE, W.J. (1973) Circulating lymphocyte subpopulation in Hashimoto's thyroiditis. Clin. exp. Immunol. 15, 345. VAN HERLE, A.J., ULLER, R.P. & MATTHEws, N.L. (1973) Radioimmunoassay for measurement of thyroglobulin in human serumj. clin. Invest. 52, 1320. WARNER, N.L. (1974) Antigen receptors on T and B lymphocytes. Adv. Immunol. 19, 67. WAMuEN, L. (1959) The thiobarbituric acid assay of sialic acid.J. biol. Chem. 234,1971. WEIGLE, W.O. (1973) Immunological unresponsiveness. Advances in Immunology (ed. F.J. Dixon and H.G. Kunkel) Vol. 16, p. 61. Academic Press, New York. WIcK, G. & RICHTER, E. (1976) Active and passive thyroglobulin rosette forming cells in obese strain (Os) chickens with spontaneous autoimmune thyroiditis. Leukocyte Membrane Determinants Regulating Immune Reactivity (eds V.P. Eijsvogel, D. Roos and W.P. Zeijlemaker). p. 229. Academic Press, New York.
Receptors for fluoresceinated human thyroglobulin in peripheral blood lymphocytes G. B. SALABE, H.
SALABfE,
L. ACCINNI & R. DOMINICI Centro di Fisiopatologia Tiroidea CNR, Clinica Medica II, Universitd di Roma, Rome, Italy
(Received 15 May 1977)
SUMMARY
Fluoresceinated human native and desialylated thyroglobulin were incubated with peripheral blood lymphocytes. 1 % of the lymphocytes, in twenty samples from normal human blood donors, showed a bright granular fluorescence where neither the number nor pattern of fluorescence differed from lymphocytes from the blood of thyroiditis patients. Fluoresceinated albumin and y-globulin did not bind to the lymphocytes, and a 500-fold excess of native non-fluoresceinated thyroglobulin inhibited the binding and pre-incubation with anti-IgM serum abolished it. Binding with desialylated thyroglobulin was negligible, and the pattern of fluorescence was pale and uniform. Analysis by sucrose gradient centrifugation and double diffusion in agar gel showed that fluorescein dissociates thyroglobulin into 12S fragments and reduces its immunoreaction with autoantibodies. It can therefore be concluded that the 12S molecule produced by fluoresceination maintains its determinants for lymphocyte receptors, whereas further dissociation, as in desialylated fluoresceinated thyroglobulin, leads to a marked reduction in the binding with lymphocytes. INTRODUCTION Antigen-binding cells identified as some of the circulating lymphocytes have been considered as an expression of the immunological status. In fact, immunization in several animal species increases the number of antigen-binding cells, and the depletion of specific antigen-binding lymphocytes abolishes the immune response towards specific antigens. Tolerant antigens, on the other hand, have a negligible number of binding lymphocytes (Ada & Cooper, 1971; Warner, 1974). The binding of thyroglobulin to peripheral (Bankhurst, Torrigiani & Allison, 1973; Roberts, Whitting & Mackay, 1973), spleen (Clagett & Weigle, 1974) and thymus (Roberts et al., 1973) lymphocytes has been revealed by the autoradiographic technique in the mouse and human (Bankhurst et al., 1973; Urbaniak, Penhale & Irvine, 1973) with highly labelled (10 pCi/,ug) 125I-labelled thyroglobulin. Binding cells were identified as B lymphocytes (Bankhurst et al., 1973; Clagett & Weigle, 1974). In mice, immunization with autologous or heterologous thyroglobulin produces an increase of thyroglobulin-binding lymphocytes (Clagett & Weigle, 1974). In patients with chronic autoimmune thyroiditis, thyroglobulin-binding lymphocytes in the peripheral blood were seen to be either increased (Roberts et al., 1973), or unchanged with respect to normal subjects (Urbaniak et al., 1973). Following immunization with homologous thyroglobulin, the strains of rats with a high susceptibility to thyroiditis do not show a larger number of thyroglobulin-binding cells than in non-susceptible strains (Penhale etal., 1975). Correspondence: Dr G. B. Salabe, Centro di Fisiopatologia Tiroidea CNR, Clinica Medica II, Universita di Roma, Rome, Italy. 0099-9140/78/0400-0159$02.00 @) 1978 Blackwell Scientific Publications
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These investigations on thyroglobulin-binding lymphocytes have been carried out so far by autoradiography with 125I-labelled thyroglobulin. No report has yet appeared on the study of thyroglobulin binding to peripheral lymphocytes using fluorescence techniques. In the present paper, the number, specificity, nature of lymphocyte receptors and the binding to fluoresceinated thyroglobulin were studied in normal and thyroiditis patients. Furthermore, chemical and immunochemical properties of the fluoresceinated binding antigen, using sucrose gradient analysis and double diffusion in agar gel, which have not been reported in studies carried out with iodinated antigen, will be described. The binding of desialylated thyroglobulin to peripheral lymphocytes has been investigated, in order to find the receptors for antigenic determinants masked by sialic acid on the lymphocytes; as was previously found in the serum antibodies from thyroiditis patients (Salabe et aL, 1976b). MATERIALS AND METHODS Fluorescein isothiocyanate (FITC) was obtained from BDH (England); the human serum albumin, twice crystallized, from Sigma and the human gamma globulin from Behringwerke AG (Marburg-Lahn, West Germany). The Ficoll-Urovison solution was prepared by dissolving 9-239 g of Ficoll (Pharmacia, Uppsala) in 119-4 ml of distilled water and 25 ml of 58% solution of Urovison (Schering SPA). The density of the solution was 1-077 g/ml at room temperature. Hanks' solution was obtained from Difco (U.S.A.) and the thyroglobulin haemagglutination test kits from Wellcome (Beckenham, England). Rabbit anti-human thyroglobulin serum was prepared as previously described (Salabe et al., 1976b) and absorbed with 0 1 ml of normal human serum. A precipitating autoantiserum was obtained from a patient with chronic thyroiditis. Highly specific anti-human y and [± chain from rabbits were supplied by Behringwerke AG (Marburg-Lahn, West Germany). Liquemin Roche was used (20 [LI per 10 ml or 10 u/ml of whole blood). Fluoresceination of thyroglobulin. A single human thyroglobulin extracted from a follicular adenoma which did not concentrate iodine at the scintiscan was used throughout the experiments. Bovine thyroglobulin was prepared from a pool of beef thyroid gland harvested from adult animals from the local slaughterhouse. Thyroglobulin from a crude saline extract (2 ml/g of minced tissue) was purified by salting-out (1.4-1-8 M S04(NH4)2) followed by chromatography on Sephadex G-200 (Salabe et al. 1976b). Analysis of the purity of the preparation by analytical ultracentrifuigation (Beckmann Spinco model E) showed that 90% of the thyroglobulin had a sedimentation rate of 19S and 10% of 4-7S. Polyacrylamide gel electrophoresis (performed in a 4% gel, pH 8-9, for 90 min at room temperature with 2-5 mA per tube) showed a single band with an electrophoretic mobility identical to that of a marker thyroglobulin. Native thyroglobulin was digested with Vibrio cholera neuraminidase according to the technique previously described (Tarutani & Shulman, 1971). The sialic acid content by the thiobarbituric assay after neuraminidase digestion was undetectable (Warren, 1959). Native and desialylated thyroglobulin, human albumin and gamma globulin were fluoresceinated by incubating a 05-1% solution of the proteins in 0 5 M NaCl, with a 0-1 M bicarbonate buffer, pH 8-6, with varying amounts (20-50 ±g/mg of thyroglobulin) of fluorescein isothiocyanate (FITC), added in powder form, for 16 hr at 4°C under gentle stirring (Coons & Kaplan, 1950; Holborow & Johnson, 1967). After incubation, the solution was extensively dialysed against phosphate buffer 0-02 M and KC 0 1 M, pH 7-4, until the diffusate no longer adsorbed at 492 nm. The concentration offluorescein in the solution ([Lg/ml) was calculated using the formula: A492 -(A320/2). fluorescein (j±g/ml) = 0-20 The concentration of protein in the solution was measured with the method of Lowry et al. (1951) or by the biuret technique (Gornal, Bardowill & David, 1949), using human albumin in the standard curve. The content of fluorescein in the protein varied from 5-15 [Lg/mg. Iodination of thyroglobulin. The iodination of the thyroglobulin was carried out with lactoperoxidase and glucose-glucose oxidase as the H202-generating system, as previously described (Pommier, Deme & Nunez, 1973; Salabe et al., 1976b). The incubation mixture contained 2 x 10-9 mol of KI, 1 mCi of 125I, carrier-free, 10-9 mol of thyroglobulin, 750 ,ug of glucose, 2 ,ug of lactoperoxidase and 2 ,ug of glucose oxidase per ml of solution. The specific activity of the labelled thyroglobulin was 2-5 mCi/mg; 0 5-1 atom of iodine per molecule of thyroglobulin. The labelled thyroglobulin was analysed by sucrose gradient centrifugation, in order to evaluate the extent of denaturation. Preparation of lymphocytes. Lymphocytes were prepared according to the method of Boyum (1968), with minor modifications. Human lymphocytes were from young blood donors at the transfusion unit or from thyroiditis patients seen at the out-patient department. The diagnosis of thyroiditis was established on the basis of a firm lobulated goitre and a high titre of anti-thyroglobulin antibodies (tanned red cell haemagglutination with a serum dilution of 1: 25 x 106). In one case, diagnosis was also confirmed by biopsy. Horse or bovine lymphocytes were prepared from blood harvested from the local slaughterhouse. 6 ml of heparinized blood were layered over 2-5 ml of Ficoll-Urovison (p = 1-077 kg/m3) in a 10 ml conical glass tube and centrifuged (Martin Christ IKS) at 1000 g at 4°C for 30 min. Before layering human blood was diluted 1: 3 with Hanks'
Receptorsforfluoresceinated human thyroglobulin
161
solution, horse blood was diluted 1: 1 and beef blood was used undiluted. At the interfaces between the Hanks' solution and Ficoll-Urovison, an opalescent ring of lymphocytes was formed, whereas red blood cells and macrophages sedimented to the bottom of the tube. The plasma and Hanks' solution were removed, the opalescent ring was collected and washed twice with Hanks' solution (7-8 ml) and once with phosphate-buffered saline (PBS) (KH2PO4-Na2HPO4, 015 M, pH 7 4) in the presence of sodium azide, 0-1%. After each wash, the solution was centrifuged at 150 g at 40 for 15 min. After the final wash the lymphocytes were suspended in PBS,with 0-1% sodium azide at a concentration of 107 per ml. The yield of lymphocytes was calculated in a Thomas-Zeiss chamber. The cells, stained by 0-01% methyl violet in citric acid, 0 1 M, were counted in both the whole blood and in the lymphocyte preparation; the yield was 15-20 x 106 lymphocytes per 10 ml of blood. The purity of the lymphocytes was evaluated in the preparation smeared by the cytocentrifuge (cytocentrifuge, Shandon) and stained with Giesma-May-Grunwald; macrophage contamination was less than 1%. The viability test was performed by incubating 1: 1 volumes of trypan blue and the lymphocytes suspension for 10 min at room temperature. Lymphocyte incubation. From 20 to 200 ±g of fluoresceinated proteins were incubated in 0-2 ml of PBS with 2 x 106 lymphocytes at 0C and in the presence of 0- 1% sodium azide. At the end of the incubation period (30 min), the lymphocytes were washed three times in 0-2 ml of PBS with sodium azide, to remove the excess of fluoresceinated unbound proteins. After each wash, the lymphocytes were run for 10 min at 150g. The inhibition of binding was performed by pre-incubation of the lymphocytes for 30 min at 4VC with rabbit anti-human IgM (0.5 ml per 5 x 106 lymphocytes) or an excess of cold, native and desialylated thyroglobulin (5-50 mg). Following the addition of the fluoresceinated protein, the mixture was incubated for an additional 30 min and washed as previously described. After the final wash, the pellet was suspended in 0-2 ml PBS with 0-1 % sodium azide. In the experiments aimed at observing the capping, the lymphocytes were incubated without sodium azide at 370C for periods offrom 10 min up to 12 hr. The lymphocytes were observed and counted under covered slides, both by fluorescence with incident light and by light microscopy, and the percentage of fluorescent cells evaluated. 2000 cells were counted for each sample with a cell counter (Clay-Adams); monocytes and cell clumps were avoided. Sucrose gradient ultracentrifugation and immunochemical properties of native and desialylated thyroglobulin. The double diffusion in agar gel was performed in 1% agar 0 04 M phosphate buffer, pH 7-1. Agar was added with 3% polyethylene glycol. Precipitin lines were stained with bromophenol blue. The sedimentation properties were analysed by linear sucrose gradient centrifugation (Spinco model L2, rotor SW27), with gradients of 5-28% or 10-40% at 26,000 rev/min at 220C for 12 hr, or 20C for 24 hr. Thirty 1 ml fractions were collected. Each fraction was estimated at 280 and 492 nm, and the "251-labelled thyroglobulin, run as the internal marker, was evaluated in a well-type scintillation counter.
RESULTS Physical and immunochemicalproperties offluoresceinated thyroglobulin The sucrose gradient centrifugation profile (Fig. 1) showed that upon fluoresceination native thyroglobulin changes its sedimentation properties: most of the fluorescence in the gradient is found in the 12S region, with only 10% remaining in the 19S region and the rest migrating with a sedimentation lower than 12S. The preparation shown in Fig. 1 had a ratio of fluorescein: protein of 15 pg/mg. At a lower level of fluoresceination (ratio fluorescein: protein of 6 jig/mg), the 19S sedimentation properties of thyroglobulin were maintained. Bovine thyroglobulin was fluoresceinated with a molar ratio of 6 and behaved as a 19S fragment in the sucrose gradient. The sucrose gradient of thyroglobulin fluoresceinated after desialylation with a ratio of 15 jig/mg revealed that the sedimentation properties of this protein were remarkably affected, and most of the absorbancy at 492 nm could not be identified in a peak and remained at the top of the gradient. Experiments were also carried out by double diffusion in agar gel to investigate the immunochemical properties of the fluoresceinated thyroglobulin (ratio of fluorescein: protein of 15 jg/mg). With rabbit anti-native human thyroglobulin antiserum (Fig. 2a), native and fluoresceinated thyroglobulin reacted identically, whereas only partial identity was seen with desialylated fluoresceinated thyroglobulin: precipitin line of native spurred over desialylated fluoresceinated thyroglobulin. With a human antithyroglobulin serum (Fig. 2b), only a faint line with fluoresceinated thyroglobulin was shown, and it did not precipitate in agar with fluoresceinated desialylated thyroglobulin. Bovine native and fluoresceinated thyroglobulin (ratioof fluorescein: protein of 6 jg/mg) reacts identically with rabbit anti-native bovine thyroglobulin. T.
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05
0*4
Cj 0~) ~0 .11
03 C 5x
.E
02
11-
a)
0-
15 10 Tube number
FIG. 1. Sucrose gradient ultracentrifugation pattern of fluoresceinated native and desialylated human thyroglobulin (SW27 rotor, gradient 10-40%, 26,000 rev/min at 220C for 12 hr). Samples were run in separate tubes of the same rotor, fractions of 1 ml were collected. Sedimentation rates were estimated by an internal 1251 marker formed by 19S and 12S human thyroglobulin labelled with lactoperoxidase. (A) 19S thyroglobulin: radioactivity vs tube number. (0) Absorbancy of desialylated thyroglobulin vs tube number; (0) absorbancy ofnative thyroglobulin vs tube number.
FIG. 2. Double diffusion in agar gel (0-04 m phospate, pH 7-4) between anti-thyroglobulin antisera and native and fluoresceinated thyroglobulins. (a) Rabbit antiserum to human 19S thyroglobulin (R) and (b) human thyroiditis serum (H) was tested with 0-1% solution of native (1), native fluoresceinated (2) and desialylated fluoresceinated thyroglobulin (3).
Number, pattern and species specificity of the binding offluoresceinated thyroglobulins to lymphocytes The number of lymphocytes binding thyroglobulin appeared to vary considerably. In twelve samples from normal blood with the same thyroglobulin preparation, fluoresceinated with 15 jig of fluorescein per mg of thyroglobulin and incubated at a concentration of 04 mg of thyroglobulin per 101 lymphocytes, the number ranged between 0-5 and 2% lymphocytes, the average being 0-99±0O4. In four thyroiditis samples examined under the same conditions, the values ranged between O'5 and 1~4%, with an average
Receptorsforfluoresceinated human thyroglobulin
163
FIG. 3. Peripheral blood lymphocytes from normal subjects incubated with fluoresceinated thyroglobulin. Two types of staining are visible at the surface of the lymphocyte: (a) granular and (b) rim. (Magnification x 800.)
of 0 9404. The number of thyroglobulin-binding lymphocytes increased with an increase in the thyroglobulin concentration (Table 1) and approached a plateau at 2 mg/ml. Below 0 05 mg/ml, the fluorescent lymphocytes were difficult to evaluate. The number of thyroglobulin-binding lymphocytes decreased and the fluorescence became pale when the cells were incubated with thyroglobulin labelled with 6 jg of fluorescein per mg of protein, instead of 15 g/ml. The most common pattern of fluorescence observed was the granular type, in which bright spots of various size and number were seen on a pale background (Fig. 3a). More rarely, the fluorescence was more diffuse on the cell surface, with a rim pattern (Fig. 3b). Finally, in some preparations, patches of fluorescence were observed on the cell. Incubation at 370C without sodium azide for 30 min to 12 hr did not produce capping of the fluorescence: there was, instead, a decrease in the number of granules on the fluorescent cells. Increasing the concentration of fluoresceinated antigens from 0 1 to 2 mg/ml for 107 lymphocytes did not change the pattern of fluorescence. Incubation of lymphocytes with desialylated thyroglobulin showed that this protein binds fewer lymphocytes than the control, and the fluorescence became pale and uniform (Table 1). TABLE 1. Effect of antigen concentration on the number of thyro-
globulin-binding lymphocytes
Fluoresceinated thyroglobulin
Native thyroglobulin-binding lymphocytes
Desialylated thyroglobulin-binding lymphocytes
(mg/ml)
(%)
(%)
0*1
13 3-3 50
0-6
05 2-0
0*7 0-6
The same preparation of human lymphocytes was subdivided into six aliquots of 2 x 106 lymphocytes, each being incubated in 0-2 ml of PBS plus 0-1% sodium azide with 0 1-2 mg of fluoresceinated thyroglobulin (15 [Lg of fluorescein per mg of thyroglobulin). After 30 min, the cells were washed and observed under a covered slide under the fluorescence microscope.
G. B. Salabel et ad.
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TABLE 2. Binding of human lymphocytes to different fluoresceinated proteins
Fluorescein Protein (and amount in mg/ml)
Human thyroglobulin (0 1) Bovine thyroglobulin (1 0) Human albumin (2-3) Human y-globulin (0-15) Human thyroglobulin (native) (50) Human thyroglobulin plus anti-IgM (0 5 ml) (0 1)
(Gg/ml)
Thyroglobulin-binding lymphocytes (%)
15 39 11-4 16 0 15
2-8 0-8 0-01 0-0-1 0-6 0
Fluoresceinated antigens were incubated for 30 min at 00C in 0-2 ml of PBS containing 0-1% sodium azide and 2-5 x 106 lymphocyte. After incubation, the cells were washed and observed under a covered slide under the fluorescent microscope. The amounts of fluorescein incorporated in each protein was variable, therefore higher concentrations of bovine thyroglobulin and human albumin, which had a lower content of fluorescein, were added to the incubation mixture. In the inhibition experiments, native human thyroglobulin was added before fluoresceinated thyroglobulin.
Experiments designed to establish the specificity of the interaction between lymphocytes and fluoresceinated human thyroglobulin are shown in Table 2. Bovine thyroglobulin bound to a lesser extent than human thyroglobulin, whereas human albumin and gamma globulin showed negligible binding. Inhibition of binding occurred only with a 500-fold excess of native thyroglobulin. The amount of each protein added was varied, in order to keep the fluorescein concentration in the incubation mixture constant. Pre-incubation of lymphocytes with rabbit anti-human IgM serum abolished the binding with fluoresceinated thyroglobulin in both normal and thyroiditis serum. The same preparation of human thyroglobulin (15 jig of fluorescein per mg) employed in the previous experiments was used to compare the binding of human lymphocytes with horse and bovine lymphocytes (Table 3). As expected, the number of thyroglobulin-binding lymphocytes increased as the thyroglobulin concentration in the incubation medium was increased. The number of thyroglobulin-binding lymphocytes was higher with human than with horse or bovine lymphocytes. In contrast, bovine thyroglobulin (6 jig of fluorescein per mg), which is 19S, bound only to heterologous (horse and human) but not to homologous lymphocytes. TABLE 3. Species specificity of lymphocytes binding thyroglo-
bulin Lymphocytes (%)
Thyroglobulin (mg/ml) Human 0.1
0-3-0-5 10 Bovine 0.3 1-15
Human
Horse
Bovine
1-3
0-8 1-2 2-6
04 0-7
3.3 5.0
-
n.d. n.d. 0-8 Lymphocytes (2 x 106) were incubated in PBS in 0-2 ml for 30 min at 0°C in 0-1% sodium azide. Human thyroglobulin was mostly dissociated as 12S (50%) whereas bovine kept its sedimentation rate of 19S. Fluorescein: protein ratio (,ug/mg) was 15 for the human and 6 for the bovine thyroglobulin. n.d. = Not detectable, (-) not done. -
0-8
165
Receptors forfluoresceinated human thyroglobulin TABLE 4. Numbers and conditions of incubation of the thyroglobulin-binding lymphocytes
Thyroglobulin* (tag/ml) ([±Ci/jg)
Lymphocytes (x 106 per ml)
0-2-025
75-91
10
0-2-0-25
28-116
10
1-2 (microsomes) 1-2 0-5-1
1
10
1 10-15
10 10
100
6-11
50
100 0-5-2-5
6-11 33-85
50 100
0 5-2 5
33-85
100
10(§
10
Source of
lymphocytes Total peripheral blood (rat) Total peripheral blood
Thyroglobulin binding lymphocytes (%) Normal Thyroiditic -
0-08 (human) 0035 Total peripheral blood (human) Thyroid (human) 0-02 Total peripheral blood (human) 0 34 Total peripheral blood (human) 20-0-5: Thymus (human) Thymus (mouse) < 0.001 (mouse), 0 03 (bovine) 0-068 (mouse), Spleen (mouse) 0-038 (bovine), 0-13 (human) 09 Total peripheral blood (human)
References
0-2-05
Penhale et al. (1975)
0-08
Urbaniak et al. (1973)
0 11
Khalid et al. (1976)
n.d.t
Khalid et al. (1976) Bankhurst et al. (1973)
2-3
Roberts et al. (1973) Roberts et al. (1973) Clagett & Weigle (1974)
0 34
Clagett & Weigle (1974)
09
Salabe et al. (1976a)
* Unless otherwise specified, the lymphocytes were incubated with homologous thyroglobulin. t Not detectable. t At birth and in the adult respectively. § Flourescein labelled (15 Lg/mg)
Better inhibition of the fluorescence was obtained with desialylated thyroglobulin. In one experiment, pre-incubation of the lymphocytes with 4 mg of native thyroglobulin was ineffective in reducing the number of fluoresceinated lymphocytes (1.5%), whereas after pre-incubation with desialylated thyroglobulin the binding of fluoresceinated native thyroglobulin with the lymphocytes was completely abolished. Sucrose gradient centrifugation of this particular preparation showed that desialylated thyroglobulin was dissociated and composed of 20% 19S and 50% 12S, a pattern similar to that of native fluoresceinated thyroglobulin.
DISCUSSION The present observations confirm the findings of Bankhurst et al. (1973) and Clagett & Weigle (1974) on the existence of a specific thyroglobulin receptor on lymphocytes. However, the number of thyroglobulin-binding lymphocytes in our experiments is ten to fifty times higher. This difference could be due to the different techniques employed (Table 4). For instance, the autoradiographic technique used by Bankhurst et al. (1973) and Clagett & Weigle (1974) gave lower estimates since the concentration of antigen in the incubation medium was 40-200 times lower than in our experiments, thus selecting only the more avid receptor. Alternatively, the receptor determinant of thyroglobulin may be damaged during 1251 iodination. When the same thyroglobulin concentrations were employed (0.10 mg/ml), a comparable number of thyroglobulin-binding lymphocytes were found (Roberts et al., 1973). The number of thyroglobulin-binding lymphocytes was highly variable. Taking into account that a standardized procedure was used in the experiments and that the cells were always counted by the same observer, and, furthermore, that the same preparation of lymphocytes repeatedly counted gave
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reproducible results, it is thus possible that the number of thyroglobulin receptor-carrying lymphocytes has a considerable individual variability. Cytophilic antibodies did not seem to increase the number of positive lymphocytes, since the average number of thyroglobulin-binding lymphocytes did not differ between normal and thyroiditis subjects in which there was a very high titre of anti-thyroglobulin antibodies. All normal subjects examined in our study had negative anti-thyroglobulin antibodies according to the TRC test; however, small quantities of circulating anti-thyroglobulin antibodies may not be detectable by TRC, therefore the possibility still existed that the number of thyroglobulin-binding lymphocytes in our experiments was increased by cytophylic antibodies. In unpublished experiments performed in our laboratory, we were unable to demonstrate any fluorescent lymphocytes after incubation of the peripheral normal lymphocytes with fluoresceinated human gamma globulin purified from serum with high titres of anti-thyroglobulin antibody. This finding limits the hypothesis that cytophylic antibodies play a role in the binding of thyroglobulin to the peripheral lymphocytes. Antigen-binding cells amongst circulating lymphocytes have been identified as an expression of the immunological status, which was demonstrated by the fact that immunization in several animal species increased the number of antigen-binding cells (Davie, Rosenthal & Paul, 1971) and the depletion of specific antigen-binding lymphocytes abolishes the immune response towards specific antigens (Ada & Byrt, 1969). With heterologous thyroglobulin an increase in antigen-binding cells was observed in mice after immunization (Clagett & Weigle, 1974). However, in rats immunized with homologous thyroglobulin in CFA, thyroiditis-susceptible strains exhibited the same number of antigen-binding lymphocytes as the non-susceptible strains (Penhale et al., 1975). Furthermore, in man, it was not possible in the present or previous studies (Urbaniak et al., 1973) to demonstrate an increase in the thyroglobulin-binding lymphocytes in the blood of patients with chronic autoimmune thyroiditis. Even lymphocytes extracted from a thyroiditic thyroid gland showed a decreased binding to thyroid microsomal antigen (Khalid, Hamilton & Cauchin, 1976). Roberts et al. (1973), on the other hand, found an increase in the thyroglobulin-binding lymphocytes. It might be suggested that in thyroiditis patients the excess of thyroglobulin or other thyroid antigens, circulating or in the thyroid, might block the receptors. An increase in circulating thyroglobulin has been demonstrated in thyroid autoimmune diseases, such as thyroiditis & Graves' disease (Van Herle, Uller & Matthews, 1973). A marked decrease in the number of binding lymphocytes has been observed with desialylated thyroglobulin. The effect of desialylation on the number of thyroglobulin-binding lymphocytes might be due to the sialic acid per se, or to the denaturation of determinants for receptors produced by fluoresceination on desialylated thyroglobulin. This latter interpretation seems to be the more likely, as the immunochemical properties of fluoresceinated desialylated thyroglobulin were altered in immunodiffusion, and previous studies have shown that the removal of sialic acid does not change the immunoreaction with circulating autoantibodies, but in some cases it enhances it, suggesting that sialic acid masks the antigenic determinants (Salabe et al. 1976b). In the present study, attention was focused on the physical properties of thyroglobulin binding to the lymphocytes. Analysis of the fluoresceinated thyroglobulin revealed that a large proportion of this antigen dissociated as a 12S fragment. It seems therefore that the 12S subunits maintains the determinants for the lymphocyte receptors, although some of the determinants for antibodies were damaged, as demonstrated by the lack of immunoprecipitation with autoantibodies in double diffusion in agar gel. Specific determinants for lymphocyte receptors on the 12S subunits are supported by indirect evidence, i.e. bovine lymphocytes do not bind to bovine thyroglobulin, which by sucrose gradient centrifugation was showed to be 19S. Non-fluoresceinated 19S thyroglobulin inhibits fluorescence at a very high concentration, whereas desialylated thyroglobulin-which is mostly dissociated as 12S fragment-inhibits the fluorescence at low concentrations. These preliminary observations might lead to further experiments aimed at establishing specific determinants on the thyroglobulin molecule. Experiments since carried out in this laboratory with purified isolated 19S and 12S thyroglobulin labelled with 1251 showed that both molecules bind with lymphocytes to the same extent, and therefore it can be concluded that
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receptors on the lymphocytes bind to determinants exposed on native 19S or dissociated in 12S thyroglobulin. Denaturation, as occurs with desialylated thyroglobulin, abolishes the reaction of receptors for thyroglobulin. According to the current view (Allison, 1973; Weigle, 1973) on the production of autoimmunity at the lymphocyte receptor level, these antigens which, like thyroglobulins, circulate at low concentration in normal subjects carry determinants specific for receptors on the B cells but not on the T cells, which are made unresponsive by the low concentration of circulating antigen. T lymphocytes become responsive when a heterologous determinant emerges from a normal constituent and reacts with specific receptors on the T cells, which in turn help or derepress the B cells and stimulate them to produce antibodies towards the normal constituent. Recent findings support this hypothesis: nude mice were unable to respond to a pool of heterologous thyroglobulin, although they possess B cells with specific receptors for mice thyroglobulin (Clagett & Weigle, 1974). However, other experiments indicate another pathway in the production of autoantibodies (de Heer & Edgington, 1976). The red blood cells of mice possess native and cryptic antigens. In the normal, animal specific receptors on B and T cells are found for the cryptic antigen whereas the receptors for native antigen are only on the B but not on the T cells. Receptors on the T cells are found only in the NZB strain, which develops haemolytic autoimmune anaemia. Furthermore, it has been found that only lymphocytes from the NZB strain produce in vitro antibodies to the native antigen (de Heer & Edgington, 1976). From these studies it emerges that autoimmunity results when a particular population of T cells is present with receptors for the native antigen. Future experiments with highly purified native thyroglobulin tested with isolated B and T cells will show whether T receptors for native thyroglobulin are peculiar only to thyroiditis patients. In our experiments, it was demonstrated that the thyroglobulin receptor binds to the native antigen. Future experiments with B and T cells purified from the blood of thyroiditis patients and native thyroid antigens will contribute to clarify which of the two proposed mechanisms is operating in thyroiditis: the appearance of a heterologous determinant on a thyroid antigen or a T-cell subpopulation with receptors for the native thyroid antigen. However, the results of preliminary experiments (Wick & Richter, 1976) have recently shown that in the spleen of obese chickens with spontaneous autoimmune thyroiditis only B lymphocytes with receptors for thyroglobulin are present. We would like to thank Mr L. Corvo for skilful technical assistance.
REFERENCES ADA, G.L. & BYRT, P. (1969) Specific inactivation of antigen reactive cells with 125I-labelled antigen. Nature (Lond.), 222, 1291. ADA, G.L. & COOPER, M.G. (1971) Antigen binding cells in tolerance and immunity. Ann. N. Y. Acad. Sci. 181,96. ALLISON, A.C. (1973) Mechanism of tolerance and autoimmunity Ann. rheum. Dis. 32, 283. BANKHURST, A.D., TORRIGIANI, G. & ALLISON, A.C. (1973) Lymphocytes binding human thyroglobulin in healthy people and its relevance to tolerance for autoantibodies. Lancet, i, 226. BoYuM, A. (1968) Separation of lymphocytes from blood and bone marrow. Scand.]. clin. Lab. Invest. 21, Suppl. 97, 1. CLAGETT, J.A. & WEIGLE, W.O. (1974) Roles of T and B lymphocytes in the termination of unresponsiveness to autologous thyroglobulin in mice.j. exp. Med. 139, 643. COONS, A.H. & KAPLAN, M.H. (1950) Localization of antigen in tissue cells. II. Improvements in a method for the detection of antigen by means of fluorescent antibody. J. exp. Med. 91, 1. DAVIE, J.M.A., ROSENTHAL, S. & PAUL, W.E. (1971) Receptors on immunocompetent cells: specificity and
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hydrate-stripped thyroglobulin. I. Preparation and physicochemical properties of desialized thyroglobulin. Biochem. biophys. Acta (Amst.), 229, 642. URBANIAK, S.J., PENHALE, W.J. & IRVINE, W.J. (1973) Circulating lymphocyte subpopulation in Hashimoto's thyroiditis. Clin. exp. Immunol. 15, 345. VAN HERLE, A.J., ULLER, R.P. & MATTHEws, N.L. (1973) Radioimmunoassay for measurement of thyroglobulin in human serumj. clin. Invest. 52, 1320. WARNER, N.L. (1974) Antigen receptors on T and B lymphocytes. Adv. Immunol. 19, 67. WAMuEN, L. (1959) The thiobarbituric acid assay of sialic acid.J. biol. Chem. 234,1971. WEIGLE, W.O. (1973) Immunological unresponsiveness. Advances in Immunology (ed. F.J. Dixon and H.G. Kunkel) Vol. 16, p. 61. Academic Press, New York. WIcK, G. & RICHTER, E. (1976) Active and passive thyroglobulin rosette forming cells in obese strain (Os) chickens with spontaneous autoimmune thyroiditis. Leukocyte Membrane Determinants Regulating Immune Reactivity (eds V.P. Eijsvogel, D. Roos and W.P. Zeijlemaker). p. 229. Academic Press, New York.
