Clin. exp. Immunol. (1976) 23, 28-32.
Binding of thyroid microsomes by lymphocytes from patients with thyroid disease and normal subjects B. A. K. KHALID, N. T. HAMILTON & M. N. CAUCHI Department of Pathology and Immunology, Monash Medical School, and Alfred Hospital, Melbourne, Australia
(Receiked 22 April 1975)
SUMMARY
Lymphocytes that could bind 125I-labelled thyroid microsomal membranes (ABL) were present in the peripheral blood of patients with various types of thyroid disease as well as in normal healthy subjects. In patients with anti-thyroid cytoplasmic antibodies the number of ABL was about three times normal (11 +2 10-60 compared with 3-26 + 018 per 104 lymphocytes). In contrast, in patients with thyroid disease without anti-thyroid cytoplasmic antibodies, the number of antigen-binding lymphocytes was not significantly greater than in the normal controls (3 94+0 14 per 104 lymphocytes). The binding of thyroid microsomes by antigen-binding lymphocytes could be blocked by thyroid microsomes but not by thyroid mitochondrial membranes, thyroglobulin or liver microsomes.
INTRODUCTION Tissue damage in autoimmune thyroiditis is generally thought to be due to hypersensitivity reactions mediated by specific lymphocytes (Delespesse et al., 1972), although complementfixing anti-thyroid microsome antibodies cytotoxic to thyroid cells in culture, usually present in the sera of patients with autoimmune thyroiditis (Roitt et al., 1964), may also be important in causing tissue damage. The immunoglobulin long-acting thyroid stimulator (LATS) found in the sera of most patients with Graves' disease can be specifically absorbed by thyroid microsomes and is generally thought to react with antigens on the surfaces of thyroid cells (Beall et al., 1969; Benhamou-Glynn et al., 1969). Several workers demonstrated specific binding of thyroglobulin by lymphocytes from the peripheral blood of patients with thyroid diseases (Bankhurst et al., 1973; Roberts et al., 1973; Urbaniak et al., 1973). This study shows that antigen-binding lymphocytes (ABL) capable of binding specifically with thyroid microsomes were present in healthy subjects and in increased numbers in thyroid disease patients with positive cytoplasmic autoantibodies. MATERIALS AND METHODS Subjects studied. Twenty-nine patients with various thyroid diseases, mostly confirmed histologically, were studied. These consisted of: patients with hyperthyroidism (thirteen, including four cases of toxic nodular goitre), thyroiditis (seven, comprising six Hashimoto's and one Reidel's disease), non-toxic goitre (five) and thyroid carcinoma (four). Fourteen normal healthy subjects of comparable age and sex were investigated. Eleven of the twenty-nine patients with thyroid disease and none of the normal subjects had autoantibodies reacting against thyroid cell cytoplasm or thyroglobulin: none had autoantibodies reacting with nuclei, mitochondria or smooth muscle. Correspondence: Mr M. N. Cauchi, Department of Pathology and Immunology, Monash Medical School, Commercial Road, Prahan, Victoria, Australia 3181.
28
Lymphocytes binding thyroid microsomes
29
Autoantibody tests. Autoantibodies were detected by indirect immunofluorescence using unfixed frozen sections (Nairn, 1975; Tannenberg et al., 1973). The Wellcome thyroglobulin haemagglutination test kit was used for anti-thyroglobulin antibodies. Lymphocytes. Lymphocytes were isolated from heparinized venous blood using Hypaque-Ficoll (Boyum, 1968). They were washed twice with ice-cold phosphate-buffered saline (PBS) containing 10% foetal calf serum (FCS) and 014% sodium azide, and then were suspended and adjusted to 5 x 106 lymphocytes in 0 5 ml of the solution. Viability as measured by Trypan Blue dye exclusion was more than 98%. To obtain thyroid lymphocytes, a surgically removed fresh thyroid gland showing severe thyroiditis was teased out and the lymphocytes, isolated from the tissue cell suspension by the same method as for peripheral blood. They were about 90% pure and more than 95% viable. Thyroid membranes. These were prepared by a modification of the method of Bingham & Burke (1972). Cells from fresh thyroids removed surgically for thyrotoxicosis, were isolated by homogenization in a tightfitting glass Dounce homogenizer until about 90% were disrupted and appeared as cell ghosts by phasecontrast microscopy. The thyroid microsomes, obtained by differential centrifugation at 105,000 g, were washed, dialysed and then further purified by centrifuging at 85,000 g through a Dextran T40 gradient consisting of a continuous gradient of 5-25% (w/w) dextran layered on top of 40Y% (w/w) dextran solution in Tris buffer. The microsomal membranes were obtained at the interface of the 25-400 concentrations. By electron microscopy these membranes appear as rough or smooth endoplasmic reticulum in vesicles some of which were attached to one another. The protein content was estimated by the method of Lowry et al. (1951) with bovine serum albumin as reference protein. The antigenicity of the membranes were determined by their ability to absorb thyroid antibodies from the sera of patients with Hashimoto disease: autoantibodies to thyroid cytoplasm and cell membranes, but not thyroglobulin, were neutralized. The microsomes were iodinated following the method of Byrt & Ada (1969) using Na 1251 (Amersham, Radiochemical Centre). The specific radioactivity of the '251-labelled microsomes was about 1-0 pCi/ug protein. Liver microsomal membranes were prepared by the same method as the thyroid microsomes. Thyroglobulin was prepared by the method of Rose & Stylos (1969). Labelling of lymphocytes with 125I-labelled thyroid microsomes. 5 x 10' lymphocytes were incubated with 600 ng of '251-labelled microsomes for 30 min at 0-4' in a final volume of 0-5 ml PBS with 10% foetal calf serum (FCS) and 0 I0 sodium azide (Byrt & Ada, 1969). The cells were washed four times by layering on a 6 0 ml gradient of PBS-FCS (100% FCS at the bottom, 75%/ in the middle, and 50% at the top) and centrifuging at I00 g for I0 min at 40C. The lymphocytes were then resuspended in 0 I ml of 100I/O FCS and smeared onto gelatin-coated slides, air-dried, fixed in methanol: water: acetic acid (89: 10: 1 v/v) and washed in distilled water. In blocking experiments, lymphocytes were first incubated for 1 hr at 4 C with the materials used for the blocking, before being washed with the PBS-FCS solution, and then they were incubated with 1251-labelled microsomes. Autoradiography. Autoradiographs made from the prepared slides by dipping into Kodak NTB-2 nuclear track emulsion, were packed into light-proof boxes and stored at 40C for 10-14 days before developing with Kodak D 19 developer, and fixing in Ilford Hypam Fixer. They were stained with Giemsa R66. An average of 10,000 cells were counted from each slide, and five slides were prepared for each antigen-binding test. Most of the positive lymphocytes had marked radioactivity at one part of the cell surface and were easily distinguishable from negative lymphocytes, damaged cells or cell debris with non-specific labelling. Positive cells in contact with each other or with debris were not counted. Average background grain count was 0-3 grains per cell area.
RESULTS All twenty-nine patients with various thyroid disease and thirteen out of fourteen normal subjects had ABL in their peripheral blood (Fig. 1). The number of ABL in patients with thyroid cytoplasmic antibodies was 11 -21 + 060 per IO0 lymphocytes which is about three times higher than in patients without autoantibodies (3-49 + 0-14 ABL per 104 lymphocytes) or normal subjects (3-26+0-18 per 104 lymphocytes). There was no significant difference between the number of ABL in patients with no autoantibodies compared with normal subjects. The ABL binding thyroid microsomes could be correlated only with the presence of thyroid cytoplasmic antibodies and not with other autoantibodies such as anti-thyroglobulin, anti-mitochondrial or anti-gastric parietal cell antibodies. There was no correlation between the titre of the anti-thyroid cytoplasmic antibodies and the number of ABL.
30
B. A. K. Khalid, N. T. Hamilton and M. N. Cauchi
No ABL could be demonstrated in the lymphocytes from the thyroid tissue of a patient with Hashimoto's disease, whereas 9 50 ABL per 104 cells were found in the peripheral blood. The binding of '25I-labelled thyroid microsomes to peripheral blood lymphocytes could be completely blocked by preincubation of the lymphocytes with excess of unlabelled thyroid microsomes (1 -80 mg/5 x 106 lymphocytes). Furthermore, the degree of blocking of the antigen-binding appeared to be proportional to the amount of unlabelled antigen used when 0-01-0-60 mg of thyroid microsome/5 x 106 lymphocytes were used (Table 1). In contrast, the binding of thyroid microsomes was unaffected by prior incubation with excess liver microsomes, thyroid mitochondrial membranes or thyroglobulin (Table 1). 2Oam20
0 _ 18 0
EA 6
In
en
o 16
U Eui
8
_0
4
6
A
82
A
A~~~
E A
B
C
FIG. 1. Number of lymphocytes binding I251-labelled microsomes in peripheral blood of patients with thyroid disease and normal subjects with (N) or without (A) cytoplasmic autoantibodies. (A) Patients with thyroid cytoplasmic autoantibody; (B) patients without thyroid cytoplasmic autoantibody; (C) normal subjects.
The binding of "25I-labelled thyroid microsomes by lymphocytes could be blocked almost completely by goat anti-human globulin serum, but not by normal goat serum or goat antirabbit globulin (Table 1).
DISCUSSION These studies show that lymphocytes capable of binding specifically with thyroid microsomes are present in the peripheral blood of patients with thyroid disease and in normal subjects. Other workers have shown that a comparable number of ABL can bind flagellin or haemocyanin (Byrt & Ada, 1969), synthetic polypeptides (Dunham et al., 1972), thyroglobulin (Bankhurst et al., 1973) and human encephalitogenic proteins (Yung et al., 1973). There was a significant increase (P = 0-001) in the number of ABL in the peripheral blood of patients who had thyroid cytoplasmic antibodies in their sera, while the number of ABL in patients who did not have autoantibodies was within the normal range. There is thus a correlation between the presence of the autoantibodies and the number of ABL, although there is no correlation between the antibody titre and the number of ABL. The ABL proliferation may be a response to excess antigen in the circulation. The present study also demonstrated that blocking of antigen-binding is directly related to
Lymphocytes binding thyroid microsomes
31
TABLE 1. Specificity of the binding of 'l25-labelled thyroid microsomes to peripheral blood lymphocytes
Preincubation of lymphocytes with:
Number of antigen-binding lymphocytes per 104 cells
Percentage blocking of
antigen binding (1) Unlabelled thyroid microsomes 001 mg 001 mg 0-24mg 0-36 mg 048 mg 0-60mg 1-80mg 440mg (2) Liver microsomes 533 mg (3) Thyroid mitochondrial membranes 5 mg (4) Thyroglobulin preparation 450mg (5) Goat anti-human whole globulin serum
(6) Preimmune goat serum
(7) Goat anti-rabbit whole globulin serum
Not preincubated
Preincubated
19-2 27 2-8 2-9 3-3 4-8 25 70
19 6 27 2-4 1.9 18 1-7 0 0
0 0 14 35 46 66 100 100
28
32
0
10 4
10 5
0
2-7 36 2-9 27 58 95 3-7 2-7 58 1.9 3-3 58
2-8 0 0 0 05 11 35 2-6 50 20 32 6-0
0 100
100 100 91-4 885 5 1 13-8 0 2 0
* Lymphocytes from individual patients or normal subjects were tested for antigen-binding before and after incubation with unlabelled antigen or serum.
the amount of antigen used. It is possible that the lack of reactivity of lymphocytes isolated from the thyroid of one patient with severe Hashimoto thyroiditis was due to exposure to abundant antigen in vivo, rendering the lymphocytes incapable of binding the 125I-labelled membranes in vitro. This may thus be a form of 'anergy' of the intrinsic lymphocytes close to the autoantigen in the advanced stage of the autoimmune disease (Nairn, 1973), the anergy being of the afferent phase of the immune reaction (Bach et al., 1973). This work was supported by grants from the Anti-Cancer Council of Victoria and the National Health and Medical Research Council. We thank Professor R. C. Nairn, Dr H. K. Muller and Dr H. A. Ward for advice and D. Quinn for technical assistance. REFERENCES BACH, F.H., SEGALL, M., ZIER, K.S., SONDEL, P.M., in healthy people and its relevance to tolerance for ALTER, B.J. & BACH, M.L. (1973) Cell mediated autoantigen. Lancet, i, 226. immunity. Separation of cells involved in recog- BEALL, G.N., DONIACH, D., RoiTT, I.M. & EL KABIR, nitive and destructive phases. Science, 180, 403. D.J. (1969) Inhibition of the long-acting thyroid BANKHURST, A.D., TORRIGIANI, G. & ALLISON, A.C. stimulator (LATS) by soluble thyroid fractions. J. (1973) Lymphocytes binding human thyroglobulin Lab. clin. Med. 73, 988.
32
B. A. K. Khalid, N. T. Hamilton and M. N. Cauchi
BENHAMOU-GLYNN, N., EL KABIR, D.J., RoITT, I.M. & DONIACH, D. (1969) Studies on the antigen reacting with the thyroid-stimulating immunoglobulin (LATS) in thyrotoxicosis. Immunology, 16, 187. BINGHAM, R.W. & BURKE, D.C. (1972) Isolation of plasma membrane and endoplasmic reticulum fragments from chick embryo fibroblasts. Biochim. biophys. Acta (Amst.), 274, 348. BOYUM, A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, supplement 97, 1. BYRT, P. & ADA, G.L. (1969) An in vitro reaction between labelled flagellin or haemocyanin and lymphocyte-like cells from normal animals. Immunology, 17, 503. DELESPESSE, G., BASTENIE, P.A., VAN HAELST, L. & NEVE, P. (1972) Thyroid autoimmunity. Thyroiditis and Thyroid Function (ed. by P. A. Bastenie and A. M. Ermans), pp. 39-67. Pergamon Press, New York. DUNHAM, E.K., UNANUE, E.R. & BENACERAF, B. (1972) Antigen binding and capping by lymphocytes of genetic non-responder mice. J. exp. Med. 136, 403. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. & RANDALL, R.J. (1951) Protein measurements with the folin phenol reagent. J. biol. Chem. 193, 265.
NAIRN, R.C. (1973) Lymphocyte anergy in cancer. Int. Res. Comm. Syst. med. Sci. 1, 34. NAIRN, R.C. (1975) Fluorescent Protein Tracing, 4th edn. Churchill Livingstone, Edinburgh and
London. ROBERTS, I.M., WHITTINGHAM, S. & MACKAY, I.R. (1973) Tolerance to an autoantigen-thyroglobulin. Lancet, ii, 936. ROITT, I.M., LING, N.R., DONIACH, D. & COUCHMAN, K.G. (1964) The cytoplasmic autoantigen of the human thyroid. I. Immunological and biochemical characteristics. Immunology, 7, 375. ROSE, N.R. & STYLOS, W.A. (1969) Splitting of human thyroglobulin. 1. Reduction and alkylation. Clin. exp. Immunol. 5, 129. TANNENBERG, A.E.G., MULLER, H.K., CAUCHI, M.N. & NAIRN, R.C. (1973) Incidence of autoantibodies in cancer patients. flin. exp. Immunol. 15, 153. URBANIAK, S.J., PENHALE, W.J. & IRVINE, W.J. (1973) Circulating lymphocyte subpopulations in Hashimoto thyroiditis. Clin. exp. Immunol. 15, 345. YUNG, L.L.L., DIENER, E., MCPHERSON, T.A., BARTON, M.A. & HYDE, H.A. (1973) Antigenbinding lymphocytes in normal man and guineapig to human encephalitogenic protein. J. Immunol. 110, 1383.
Binding of thyroid microsomes by lymphocytes from patients with thyroid disease and normal subjects B. A. K. KHALID, N. T. HAMILTON & M. N. CAUCHI Department of Pathology and Immunology, Monash Medical School, and Alfred Hospital, Melbourne, Australia
(Receiked 22 April 1975)
SUMMARY
Lymphocytes that could bind 125I-labelled thyroid microsomal membranes (ABL) were present in the peripheral blood of patients with various types of thyroid disease as well as in normal healthy subjects. In patients with anti-thyroid cytoplasmic antibodies the number of ABL was about three times normal (11 +2 10-60 compared with 3-26 + 018 per 104 lymphocytes). In contrast, in patients with thyroid disease without anti-thyroid cytoplasmic antibodies, the number of antigen-binding lymphocytes was not significantly greater than in the normal controls (3 94+0 14 per 104 lymphocytes). The binding of thyroid microsomes by antigen-binding lymphocytes could be blocked by thyroid microsomes but not by thyroid mitochondrial membranes, thyroglobulin or liver microsomes.
INTRODUCTION Tissue damage in autoimmune thyroiditis is generally thought to be due to hypersensitivity reactions mediated by specific lymphocytes (Delespesse et al., 1972), although complementfixing anti-thyroid microsome antibodies cytotoxic to thyroid cells in culture, usually present in the sera of patients with autoimmune thyroiditis (Roitt et al., 1964), may also be important in causing tissue damage. The immunoglobulin long-acting thyroid stimulator (LATS) found in the sera of most patients with Graves' disease can be specifically absorbed by thyroid microsomes and is generally thought to react with antigens on the surfaces of thyroid cells (Beall et al., 1969; Benhamou-Glynn et al., 1969). Several workers demonstrated specific binding of thyroglobulin by lymphocytes from the peripheral blood of patients with thyroid diseases (Bankhurst et al., 1973; Roberts et al., 1973; Urbaniak et al., 1973). This study shows that antigen-binding lymphocytes (ABL) capable of binding specifically with thyroid microsomes were present in healthy subjects and in increased numbers in thyroid disease patients with positive cytoplasmic autoantibodies. MATERIALS AND METHODS Subjects studied. Twenty-nine patients with various thyroid diseases, mostly confirmed histologically, were studied. These consisted of: patients with hyperthyroidism (thirteen, including four cases of toxic nodular goitre), thyroiditis (seven, comprising six Hashimoto's and one Reidel's disease), non-toxic goitre (five) and thyroid carcinoma (four). Fourteen normal healthy subjects of comparable age and sex were investigated. Eleven of the twenty-nine patients with thyroid disease and none of the normal subjects had autoantibodies reacting against thyroid cell cytoplasm or thyroglobulin: none had autoantibodies reacting with nuclei, mitochondria or smooth muscle. Correspondence: Mr M. N. Cauchi, Department of Pathology and Immunology, Monash Medical School, Commercial Road, Prahan, Victoria, Australia 3181.
28
Lymphocytes binding thyroid microsomes
29
Autoantibody tests. Autoantibodies were detected by indirect immunofluorescence using unfixed frozen sections (Nairn, 1975; Tannenberg et al., 1973). The Wellcome thyroglobulin haemagglutination test kit was used for anti-thyroglobulin antibodies. Lymphocytes. Lymphocytes were isolated from heparinized venous blood using Hypaque-Ficoll (Boyum, 1968). They were washed twice with ice-cold phosphate-buffered saline (PBS) containing 10% foetal calf serum (FCS) and 014% sodium azide, and then were suspended and adjusted to 5 x 106 lymphocytes in 0 5 ml of the solution. Viability as measured by Trypan Blue dye exclusion was more than 98%. To obtain thyroid lymphocytes, a surgically removed fresh thyroid gland showing severe thyroiditis was teased out and the lymphocytes, isolated from the tissue cell suspension by the same method as for peripheral blood. They were about 90% pure and more than 95% viable. Thyroid membranes. These were prepared by a modification of the method of Bingham & Burke (1972). Cells from fresh thyroids removed surgically for thyrotoxicosis, were isolated by homogenization in a tightfitting glass Dounce homogenizer until about 90% were disrupted and appeared as cell ghosts by phasecontrast microscopy. The thyroid microsomes, obtained by differential centrifugation at 105,000 g, were washed, dialysed and then further purified by centrifuging at 85,000 g through a Dextran T40 gradient consisting of a continuous gradient of 5-25% (w/w) dextran layered on top of 40Y% (w/w) dextran solution in Tris buffer. The microsomal membranes were obtained at the interface of the 25-400 concentrations. By electron microscopy these membranes appear as rough or smooth endoplasmic reticulum in vesicles some of which were attached to one another. The protein content was estimated by the method of Lowry et al. (1951) with bovine serum albumin as reference protein. The antigenicity of the membranes were determined by their ability to absorb thyroid antibodies from the sera of patients with Hashimoto disease: autoantibodies to thyroid cytoplasm and cell membranes, but not thyroglobulin, were neutralized. The microsomes were iodinated following the method of Byrt & Ada (1969) using Na 1251 (Amersham, Radiochemical Centre). The specific radioactivity of the '251-labelled microsomes was about 1-0 pCi/ug protein. Liver microsomal membranes were prepared by the same method as the thyroid microsomes. Thyroglobulin was prepared by the method of Rose & Stylos (1969). Labelling of lymphocytes with 125I-labelled thyroid microsomes. 5 x 10' lymphocytes were incubated with 600 ng of '251-labelled microsomes for 30 min at 0-4' in a final volume of 0-5 ml PBS with 10% foetal calf serum (FCS) and 0 I0 sodium azide (Byrt & Ada, 1969). The cells were washed four times by layering on a 6 0 ml gradient of PBS-FCS (100% FCS at the bottom, 75%/ in the middle, and 50% at the top) and centrifuging at I00 g for I0 min at 40C. The lymphocytes were then resuspended in 0 I ml of 100I/O FCS and smeared onto gelatin-coated slides, air-dried, fixed in methanol: water: acetic acid (89: 10: 1 v/v) and washed in distilled water. In blocking experiments, lymphocytes were first incubated for 1 hr at 4 C with the materials used for the blocking, before being washed with the PBS-FCS solution, and then they were incubated with 1251-labelled microsomes. Autoradiography. Autoradiographs made from the prepared slides by dipping into Kodak NTB-2 nuclear track emulsion, were packed into light-proof boxes and stored at 40C for 10-14 days before developing with Kodak D 19 developer, and fixing in Ilford Hypam Fixer. They were stained with Giemsa R66. An average of 10,000 cells were counted from each slide, and five slides were prepared for each antigen-binding test. Most of the positive lymphocytes had marked radioactivity at one part of the cell surface and were easily distinguishable from negative lymphocytes, damaged cells or cell debris with non-specific labelling. Positive cells in contact with each other or with debris were not counted. Average background grain count was 0-3 grains per cell area.
RESULTS All twenty-nine patients with various thyroid disease and thirteen out of fourteen normal subjects had ABL in their peripheral blood (Fig. 1). The number of ABL in patients with thyroid cytoplasmic antibodies was 11 -21 + 060 per IO0 lymphocytes which is about three times higher than in patients without autoantibodies (3-49 + 0-14 ABL per 104 lymphocytes) or normal subjects (3-26+0-18 per 104 lymphocytes). There was no significant difference between the number of ABL in patients with no autoantibodies compared with normal subjects. The ABL binding thyroid microsomes could be correlated only with the presence of thyroid cytoplasmic antibodies and not with other autoantibodies such as anti-thyroglobulin, anti-mitochondrial or anti-gastric parietal cell antibodies. There was no correlation between the titre of the anti-thyroid cytoplasmic antibodies and the number of ABL.
30
B. A. K. Khalid, N. T. Hamilton and M. N. Cauchi
No ABL could be demonstrated in the lymphocytes from the thyroid tissue of a patient with Hashimoto's disease, whereas 9 50 ABL per 104 cells were found in the peripheral blood. The binding of '25I-labelled thyroid microsomes to peripheral blood lymphocytes could be completely blocked by preincubation of the lymphocytes with excess of unlabelled thyroid microsomes (1 -80 mg/5 x 106 lymphocytes). Furthermore, the degree of blocking of the antigen-binding appeared to be proportional to the amount of unlabelled antigen used when 0-01-0-60 mg of thyroid microsome/5 x 106 lymphocytes were used (Table 1). In contrast, the binding of thyroid microsomes was unaffected by prior incubation with excess liver microsomes, thyroid mitochondrial membranes or thyroglobulin (Table 1). 2Oam20
0 _ 18 0
EA 6
In
en
o 16
U Eui
8
_0
4
6
A
82
A
A~~~
E A
B
C
FIG. 1. Number of lymphocytes binding I251-labelled microsomes in peripheral blood of patients with thyroid disease and normal subjects with (N) or without (A) cytoplasmic autoantibodies. (A) Patients with thyroid cytoplasmic autoantibody; (B) patients without thyroid cytoplasmic autoantibody; (C) normal subjects.
The binding of "25I-labelled thyroid microsomes by lymphocytes could be blocked almost completely by goat anti-human globulin serum, but not by normal goat serum or goat antirabbit globulin (Table 1).
DISCUSSION These studies show that lymphocytes capable of binding specifically with thyroid microsomes are present in the peripheral blood of patients with thyroid disease and in normal subjects. Other workers have shown that a comparable number of ABL can bind flagellin or haemocyanin (Byrt & Ada, 1969), synthetic polypeptides (Dunham et al., 1972), thyroglobulin (Bankhurst et al., 1973) and human encephalitogenic proteins (Yung et al., 1973). There was a significant increase (P = 0-001) in the number of ABL in the peripheral blood of patients who had thyroid cytoplasmic antibodies in their sera, while the number of ABL in patients who did not have autoantibodies was within the normal range. There is thus a correlation between the presence of the autoantibodies and the number of ABL, although there is no correlation between the antibody titre and the number of ABL. The ABL proliferation may be a response to excess antigen in the circulation. The present study also demonstrated that blocking of antigen-binding is directly related to
Lymphocytes binding thyroid microsomes
31
TABLE 1. Specificity of the binding of 'l25-labelled thyroid microsomes to peripheral blood lymphocytes
Preincubation of lymphocytes with:
Number of antigen-binding lymphocytes per 104 cells
Percentage blocking of
antigen binding (1) Unlabelled thyroid microsomes 001 mg 001 mg 0-24mg 0-36 mg 048 mg 0-60mg 1-80mg 440mg (2) Liver microsomes 533 mg (3) Thyroid mitochondrial membranes 5 mg (4) Thyroglobulin preparation 450mg (5) Goat anti-human whole globulin serum
(6) Preimmune goat serum
(7) Goat anti-rabbit whole globulin serum
Not preincubated
Preincubated
19-2 27 2-8 2-9 3-3 4-8 25 70
19 6 27 2-4 1.9 18 1-7 0 0
0 0 14 35 46 66 100 100
28
32
0
10 4
10 5
0
2-7 36 2-9 27 58 95 3-7 2-7 58 1.9 3-3 58
2-8 0 0 0 05 11 35 2-6 50 20 32 6-0
0 100
100 100 91-4 885 5 1 13-8 0 2 0
* Lymphocytes from individual patients or normal subjects were tested for antigen-binding before and after incubation with unlabelled antigen or serum.
the amount of antigen used. It is possible that the lack of reactivity of lymphocytes isolated from the thyroid of one patient with severe Hashimoto thyroiditis was due to exposure to abundant antigen in vivo, rendering the lymphocytes incapable of binding the 125I-labelled membranes in vitro. This may thus be a form of 'anergy' of the intrinsic lymphocytes close to the autoantigen in the advanced stage of the autoimmune disease (Nairn, 1973), the anergy being of the afferent phase of the immune reaction (Bach et al., 1973). This work was supported by grants from the Anti-Cancer Council of Victoria and the National Health and Medical Research Council. We thank Professor R. C. Nairn, Dr H. K. Muller and Dr H. A. Ward for advice and D. Quinn for technical assistance. REFERENCES BACH, F.H., SEGALL, M., ZIER, K.S., SONDEL, P.M., in healthy people and its relevance to tolerance for ALTER, B.J. & BACH, M.L. (1973) Cell mediated autoantigen. Lancet, i, 226. immunity. Separation of cells involved in recog- BEALL, G.N., DONIACH, D., RoiTT, I.M. & EL KABIR, nitive and destructive phases. Science, 180, 403. D.J. (1969) Inhibition of the long-acting thyroid BANKHURST, A.D., TORRIGIANI, G. & ALLISON, A.C. stimulator (LATS) by soluble thyroid fractions. J. (1973) Lymphocytes binding human thyroglobulin Lab. clin. Med. 73, 988.
32
B. A. K. Khalid, N. T. Hamilton and M. N. Cauchi
BENHAMOU-GLYNN, N., EL KABIR, D.J., RoITT, I.M. & DONIACH, D. (1969) Studies on the antigen reacting with the thyroid-stimulating immunoglobulin (LATS) in thyrotoxicosis. Immunology, 16, 187. BINGHAM, R.W. & BURKE, D.C. (1972) Isolation of plasma membrane and endoplasmic reticulum fragments from chick embryo fibroblasts. Biochim. biophys. Acta (Amst.), 274, 348. BOYUM, A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, supplement 97, 1. BYRT, P. & ADA, G.L. (1969) An in vitro reaction between labelled flagellin or haemocyanin and lymphocyte-like cells from normal animals. Immunology, 17, 503. DELESPESSE, G., BASTENIE, P.A., VAN HAELST, L. & NEVE, P. (1972) Thyroid autoimmunity. Thyroiditis and Thyroid Function (ed. by P. A. Bastenie and A. M. Ermans), pp. 39-67. Pergamon Press, New York. DUNHAM, E.K., UNANUE, E.R. & BENACERAF, B. (1972) Antigen binding and capping by lymphocytes of genetic non-responder mice. J. exp. Med. 136, 403. LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. & RANDALL, R.J. (1951) Protein measurements with the folin phenol reagent. J. biol. Chem. 193, 265.
NAIRN, R.C. (1973) Lymphocyte anergy in cancer. Int. Res. Comm. Syst. med. Sci. 1, 34. NAIRN, R.C. (1975) Fluorescent Protein Tracing, 4th edn. Churchill Livingstone, Edinburgh and
London. ROBERTS, I.M., WHITTINGHAM, S. & MACKAY, I.R. (1973) Tolerance to an autoantigen-thyroglobulin. Lancet, ii, 936. ROITT, I.M., LING, N.R., DONIACH, D. & COUCHMAN, K.G. (1964) The cytoplasmic autoantigen of the human thyroid. I. Immunological and biochemical characteristics. Immunology, 7, 375. ROSE, N.R. & STYLOS, W.A. (1969) Splitting of human thyroglobulin. 1. Reduction and alkylation. Clin. exp. Immunol. 5, 129. TANNENBERG, A.E.G., MULLER, H.K., CAUCHI, M.N. & NAIRN, R.C. (1973) Incidence of autoantibodies in cancer patients. flin. exp. Immunol. 15, 153. URBANIAK, S.J., PENHALE, W.J. & IRVINE, W.J. (1973) Circulating lymphocyte subpopulations in Hashimoto thyroiditis. Clin. exp. Immunol. 15, 345. YUNG, L.L.L., DIENER, E., MCPHERSON, T.A., BARTON, M.A. & HYDE, H.A. (1973) Antigenbinding lymphocytes in normal man and guineapig to human encephalitogenic protein. J. Immunol. 110, 1383.
