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Immune complex deposits in thyroid glands of patients with Graves' disease: I. Complement system in serum and thyroid gland of patients with Graves' disease
CLINICAL
IMMUNOLOGY
Immune
AND
IMMUNOPATHOLOGY
Complex
I. Complement
19, 98- 108 (1981)
Deposits in Thyroid Glands of Patients with Graves’ Disease:
System
in Serum and Thyroid Graves’ Disease
Gland of Patients
HIROSHI FUJIWARA, MOTOMICHI TORISU,’ TORU BABA,~ Division
of Clinical
immunology,
YASUSH~ AND HARUO ESAKI~
Department of First Surgery, Medicine, Fukuoka 812, Japan
Kyushu
with
KOITABASHI,* University
School
of
Received August 25, 1980 Serum complement level in 77 patients with Graves’ disease was periodically measured before and after various treatments including antithyroid drug administration and surgical operation. The complement titers showed remarkable fluctuation during the preoperative periods. However, the average complement titers in patients with this disease were not significantly different from those in patients with other thyroid diseases and in healthy volunteers. In patients whose clinical signs improved after surgical operation, the fluctuation of complement titers became smaller, falling within a normal range. To examine the possible local activation of complement system in thyroid glands of Graves’ disease, immunofluorescent stainings were performed in 30 thyroid glands with this disease utilizing fluorescein isothiocyanate (FITC)-labeled antisera against immunoglobulins and complement components. Granular deposition of IgG, IgA, IgB, Clq, C3, and C9 was observed along the follicular basement membrane (FBM) and/or at the stroma. IgG deposits along the FBM were usually accompanied with Clq, C3, and C9 deposition. None of 12 IgG-positive glands were stained by FITC-labeled antiserum to fibrinogen. Seven normal glands showed no significant staining for any of those immunoglobulins and complement components. These observations indicate that complement system may be locally activated in the thyroid gland via the classical pathway and that deposition of soluble immune complexes may occur in the FBM of Graves’ disease. Such local activation of complement may be in part the cause of strong fluctuation of complement levels in hyperthyroidism patients.
INTRODUCTION Antithyroid antibodies have been detected in patients with Graves’ disease as frequently as in those with Hashimoto’s thyroiditis. In addition, the discovery of long-acting thyroid stimulator (LATS) in Graves’ disease (1) and the subsequent demonstration that LATS is biologically and immunologically identical with im’ Address reprint requests to Motomichi Torisu, M.D., Division of Clinical Immunology, Department of First Surgery, Kyushu University School of Medicine, 3-l-l Maedashi Higashi-Ku, Fukuoka 812, Japan. * Present address: Department of Pediatrics, St. Marianna University School of Medicine, Kawasaki 213, Japan. 3 Present address: Department of Dermatology, Institute of Clinical Medicine, The University of Tsukuba, Ibaraki 300-3 1, Japan. * Present address: Department of Second Surgery, Hiroshima University School of Medicine, Hiroshima 734, Japan. 98 0090-1229/81/040098-11$01.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
COMPLEMENT
AND
GRAVES’
DISEASE
99
munoglobulin G (2, 3), prompted many investigators to perform various immunological studies on this disease (4, 5). However, its etiological details still remain unclear. The role of serum complement in the pathogenesis of systemic lupus erythematosus (SLE) (6), rheumatoid arthritis (RA) (7), and glomerulonephritis (8) was well documented. In contrast, serum complement studies in Graves’ disease and Hashimoto’s thyroiditis have been rather scarce (9, 10). In this communication, we will describe the interesting fluctuation of complement levels in patients with Graves’ disease and the evidence indicating the activation of complement system in thyroid glands of Graves’ disease. These results suggest the important role played by the complement system for the pathogenesis of Graves’ disease. MATERIALS
AND METHODS
Patients. This study was composed of 77 patients with Graves’ disease (15 males, 62 females; mean age of 34 + 12 years). These patients were confirmed by both clinical and laboratory findings, and classified.as follows according to their clinical status: (i) 12 untreated patients with hyperthyroid signs; (ii) 7 patients with the recurrent disease. They had undergone antithyroid drug therapy and/or operation. They had shown remarkable improvements clinically for several years after these treatments. However, when they visited our clinic again, they were found to have goiters and hyperthyroid symptoms; (iii) 58 patients under the treatment with antithyroid drugs (56 patients) or with the radioiodine therapy (2 patients). Two patients with radioiodine therapy had hyperthyroid signs during the current experimental period. As a control group, 57 patients with Hashimoto’s thyroiditis, 15 with welldifferentiated adenocarcinoma of the thyroid, 13 with thyroid adenoma, and 10 with adenomatous goiter were studied together with 500 healthy volunteers. Serum sampling. Approximately 10 ml of blood were obtained aseptically by venipuncture. After the blood was clotted at room temperature for approximately 1 hr, serum was separated by centrifugation at SOOgfor 10 min and stored at - 70°C until just prior to use. Serum thyroid hormone levels. Concentration of serum total thyroxin (T,) was measured by competitive protein-binding analysis using Res-O-Mat T, (Daiichi Radioisotope Laboratories, Ltd., Tokyo) (normal range, 5.0-13.7 p&/100 ml). Serum triiodothyronine (T,) was assayed by radioimmunoassay using RIA-Mat T, (Daiichi Radioisotope Laboratories, Ltd., Tokyo) (normal range, 90-210 ng/ 100 ml). Serum antithyroid antibodies. Thyroid antibodies to thyroglobulin (TGHA) and microsomes (MCHA) were measured by the tanned sheep red cell hemagglutination technique using thyroid test kit and microsome test kit, respectively (Fujizoki Pharmaceutical Co. Ltd., Tokyo). Serum complement assays. Total complement activity was measured in 50% hemolytic units (CH50) according to Mayer (11) and with the immune adherence hemagglutination (IA50) method of Nishioka (12). The activity of Cl, C4, and C2 was assayed hemolytically according to a modified method of Nelson et al. (13)
100
FUJIWARA
ET AL.
using microtiter plastic plates. The C3-C9 (C3T) activity was measured hemolytically as previously described (14). The C9 activity was titrated by immune hemolysis according to the method of Ruddy et al. (15). Zmmunochemicaf assay. Serum concentration of Clq subunit of Cl (16) and C5 (17) was determined as previously described using the single radial immunodiffusion method of Mancini et al. (18). The amount of C4 and C3 was measured using Partigen plates from Behring Institute, Marburg, West Germany. ZmmunojZuorescent studies. Thyroid tissues obtained at the time of operation or biopsy were quickly frozen in liquid nitrogen and stored at -70°C until examined. Tissue blocks were cut at 4-5 pm on a cryostat at -20°C. Fixation and immunofluorescent staining of tissue sections were performed according to the standard procedures (19). Presence of IgM, IgG, IgA, C3, and fibrinogen was demonstrated by direct immunofluorescent technique using FITC-labeled rabbit antisera (Behring Institute, Marburg, West Germany) to human IgM, IgG, IgA, C3, and fibrinogen, respectively. The demonstration of the presence of IgE, Clq, C4, C9, and C3 activator (C3A) was carried out by indirect immunofluorescent technique employing rabbit antisera to human IgE, Clq, C4, C9, and C3A (Behring Institute, Marburg, West Germany), respectively, and FITC-labeled goat antiserum to rabbit IgG (Melory Laboratories, Inc., Springfield, Va.). Statistical analysis. Experimental values in each group were analyzed by F test and Student’s t test. RESULTS Serum Complement
Activities
in Patients
with Graves’ Disease
The mean complement titers measured in patients with various thyroid diseases are shown in Table 1. The mean complement activity in Graves’ disease was 35.7 ? 8.1 CHSO/ml. No significant difference between this value and that of control patients or that of healthy volunteers was observed. However, the value of variance of the complement titers in patients with Graves’ disease and Hashimoto’s thyroiditis was significantly larger (P < 0.01) than that of healthy volunteers. The complement titers in 21 patients with Graves’ disease were periodically TABLE TOTAL COMPLEMENT ACTIVITY OF THE DISEASE, HASHIMOTO’S THYROIDITIS, No.
Diagnosis Graves’ disease Hashimoto’s thyroiditis Well-differentiated adenocarcinoma Adenoma Adenomatous goiter Healthy volunteers
1 SERA FROM PATIENTS AND OTHER THYROID
of
patients
WITH GRAVES’ DISEASES
Total complement activity (CHSO/ml; mean it 1 SD)
77 57 I5
35.7 i: 8.1 37.1 + 7.6 33.8 ” 4.4
13 10 500
36.5 +- 5.9 35.1 + 5.2 36.5 -c 5.8
COMPLEMENT
AND
GRAVES’
101
DISEASE
c9 unitlml
,o(
I Thyroid test 1#*~Microsome
3.9%
-8
4.1%
-6
-4
-2
0
2
4
test
6.7%
6
8 Time
10 12 24 36 in months
FIG. 1. Hemolytic complement titers, antithyroid antibody titers, and thyroid function tests in a patient with Graves’ disease before and after operation. Operation was performed at time zero. Hemolytic activities, especially C4 activities, were unstable during the preoperative course.
measured before and after treatments. Figures 1 and 2 show two typical cases of Graves’ disease with their various laboratory findings including complement and antithyroid antibody levels frequently measured before and after surgical operation. The changes of complement levels in.these cases were remarkably unstable during the preoperative period when serum thyroid hormone levels were elevated. However, after surgical operation, the fluctuation of complement levels became smaller, falling within a normal range. Thirteen out of twenty-one patients with this disease (62%) showed a similar fluctuation of complement levels as seen in these two cases during the preoperative period. However, the frequency of the preoperative fluctuation was not significantly different from that in healthy vol-
102
FUJIWARA
-6
-6
-4
-2
0
ET AL.
2
4
6
6 lime
10 12 24 36 in months
FIG. 2. Complement levels, antithyroid antibody titers, and thyroid function tests in a patient with Graves’ disease before and after operation. Operation was performed at time zero. Complement levels were unstable throughout the preoperative course. However, the levels became stable within normal limits after operation.
unteers. In alI 15 patients whose clinical signs improved after surgical operation, the complement titers became stable and maintained a normal level after operation. In Hashimoto’s thyroiditis, the complement levels in 6 out of 14 patients (43%) who were serially measured before and after treatment with thyroid hormone drugs showed a similar fluctuation as seen in Graves’ disease during the current experimental period. In contrast, no fluctuation of complement titers in patients with well-differentiated adenocarcinoma, adenoma, or adenomatous goiter was noticed.
COMPLEMENT
Relationship between Thyroid Graves’ Disease
AND
Hormone
GRAVES’
103
DISEASE
Levels and Complement
Activity
in
In order to explore a possible relationship between the degree of thyroid function and the complement activity in Graves’ disease, 77 patients with this disease were divided into three groups according to serum T4 level (see Materials and Methods). The mean complement titers in 24 patients with an elevated level of serum Tq, 37 patients with a normal level, and 16 patients with a low level were 37.9 + 9.2,35.7 t 7.6, and 36.3 k 7.8 CHSO/ml, respectively. No clear association existed between the thyroid hormone levels and the mean or variance of the complement titers in these groups. However, the value of standard deviation of the complement titers had a tendency to narrow with the improvement of clinical signs after appropriate treatments. Relationship between Thyroid Graves’ Disease
Antibodies
and Complement
Activity
in
The sera from 72 out of 77 patients with this disease (93%) contained thyroid antibodies (TGHA and/or MCHA) and no antibody was detected in the other five patients. The mean complement titer in patients with serum antithyroid antibodies was 36.0 2 8.0 CHSO/ml and that in the antibody-negative patients was 33.1 + 5.1 CHSO/ml. The difference of complement titers between these two groups was not statistically significant. Immunojluorescent Graves’ Disease
Studies on the Thyroid
Glands in Patients
with
The abnormal fluctuation of serum complement levels in patients with Graves’ disease as described above seems to suggest that the complement system may be involved in pathological process of this disease. Therefore, we have next explored for possible deposition of immunoglobulins and complement components in thyroid glands of Graves’ disease patients, utilizing both direct and indirect immunofluorescent staining techniques. As summarized in Table 2, granular deposition of immunoglobulins and complement components was recognized along the FBM in a significant number of glands examined; IgG deposition was found in 12 specimens (40%) out of 30 examined. Similarly detected were IgA (3%), IgE (17%), Clq (50%), C3 (20%), and C9 (88%). Immunofluorescent staining with C9 in this disease was performed only in IgG-positive thyroid specimens. In addition to the deposition along the FBM, immunoglobulins and complement components were also stained at the stroma of thyroid glands. Deposition of IgG, IgA, IgE, Clq, and C9 was detected in 27, 13, 22,58, and 13%, respectively, of the specimens examined. As also shown in Table 2, we could detect fibrinogen in 7 cases out of 24 thyroid glands examined. Interestingly, only one out of these seven specimens showed the concurrent deposition of IgE and Clq. The other six glands did not show any positive staining for immunoglobulins or complement components. Typical granular staining of IgG and C3 is demonstrated in Figs. 3 and 4, respectively. Staining pattern of IgA, IgE, Clq, C9, and fibrinogen was similar to that pictured in these figures.
104
FUJIWARA TABLE IMMUNOFLUORESCENT
STAINING
GRAVES’
DISEASE
Graves’ disease Expt
No.
Stroma
kM
30 30 30 23 12 7 30 8” 8 24
0 8 4 5 7 0 0 1 0 6
I@ Id I@ Ch c4 c3 c9 C3A Fib’
AND
ET AL. 2 OF THE
HASHIMOTO’S
THYROID
GLANDS
IN
THYROIDITIS
Hashimoto’s thyroiditis
Normal glands”
FBMb
No.
Stroma
FBM
No.
Stroma
FBM
0 12 1 4 6 0 6 7 0 1
17 17 17 17 14 10 17 9 10 12
6 14 10 8 3 2 2 3 2 10
0 3 2 2 I 1 2 2 1 0
7 7 7 1 7 NT” 7 NT NT 4
0 0 0 0 0 NT 0 NT NT 1
0 0 0 0 0 NT 0 NT NT 0
” Normal glands were obtained from the uninvolved tissue of nontoxic nodular goiter. ’ FBM: Follicular basement membrane. c Fib: Fibrinogen. ’ Immunofluorescent staining with C9 in Graves’ disease was performed only for IgG positive cases along the FBM. ( NT: Not tested
In order to compare these pathological findings with those in the well-known autoimmune thyroiditis, we have next examined thyroid glands with Hashimoto’s thyroiditis for staining of immunoglobulins and complement components. As expected, a significant number of specimens from this type of thyroiditis have shown positive staining for various immunoglobulins and complement components both along the FBM and in the stroma, in the same pattern as that of Graves’ disease. The stromal deposition of immunoglobulins and complement components except Clq was encountered more frequently in this disease than in Graves’ disease. In addition, stromal deposition of fibrinogen was recognized frequently in this disease (83%), whereas no FBM deposition of fibrinogen was found. These results are also summarized in Table 2. In contrast, none of the seven normal thyroid glands obtained from patients with nontoxic nodular goiter showed any specific staining for any types of immunoglobulins or complement components tested. These results provide stronger evidence that the complement system may have an important role in the pathological development of Graves’ disease as has been suggested by Kalderon and Bogaars for Graves’ disease and Hashimoto’s thyroiditis (20). DISCUSSION In the present communication, we have described that the serum complement activity in patients with Graves’ disease fluctuated conspicuously as well as in Hashimoto’s thyroiditis although the mean titer of the activity was not significantly different from that in other thyroid disease patients or healthy volunteers. The inability of Graves’ disease patients to maintain the complement activity at a stable level was corrected after surgical operation. These initial observations have led us to believe in the active participation of the complement system in this
COMPLEMENT
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DISEASE
105
FIG. 3. Immunofluorescence demonstration of granular deposits of IgG along the FBM in a patient with Graves’ disease. This specimen showed positive staining for IgG, IgE, Clq, C3, and C9 along the FBM and at the stroma. Original magnification, x 100.
deposits are evident along the FBM. Original magnification,
106
FUJIWARA
ET AL.
disease process. We were able to show that various types of immunoglobulins (IgG, IgA, and IgE) and complement components (Clq, C3, and C9) were deposited along the FBM and in the stroma of thyroid glands. As far as we know, this is the first demonstration of IgG, Clq, C3, and C9 being found simultaneously deposited along the FBM in Graves’ disease. Such fluctuation of complement titers as seen in Graves’ disease has been repotted in myasthenia gravis (21, 22), which thymoma or thymic hyperplasia is frequently implicated. In addition, the fluctuation of the titers in myasthenia gravis became much smaller after surgical operation and, in some cases, fell within a normal range. However, its precise mechanism still remains unclear. The decrease in the whole complement activity and early components of complement (Clq, C4, C2, and C3) have been reported in SLE, a representative autoimmune disease (23). The mechanism of this depression is presumably due to activation and subsequent consumption of the complement system by the presence of circulating immune complexes. Although circulating immune complexes have been reported in Graves’ disease (24-26), the present study has revealed that serum complement titers did not show significant changes regardless of the presence or absence of circulating thyroid antibodies. These results suggest that the complement consumption may not occur in the circulation to such an extent that serum complement titers are significantly decreased in Graves’ disease. It is obvious, however, that further investigations are required for clarification of the underlying mechanism to explain these apparently conflicting observations. On the other hand, in RA, the level of serum complement activity is normal or even elevated, while the complement titer in synovial fluids is strikingly diminished in comparison with other joint diseases (27) and immunoglobulins and complement components are found deposited in the synovial membranes (28). These findings in RA seem to indicate the importance of local activation and subsequent consumption of the complement system without much effect on the serum complement level during a certain disease process. Actually in the present study we have found the granular deposition of IgG, Clq, C3, and C9 along the FBM area and in the stroma of thyroid glands with Graves’ disease, in spite of no apparent depression of the serum complement level. In contrast, the seven normal glands obtained from patients with nontoxic nodular goiter showed no significant staining along the FBM or in the stroma for immunoglobulins and complement components tested. In the present study, IgG deposition along the FBM was usually accompanied with Clq, C3, and C9 deposition. Although deposits of immunoglobulins and C3 component in the FBM of Graves’ thyroid glands were previously reported (29, 30), this is the first demonstration that IgG, Clq, C3, and C9 were deposited together along the FBM in this disease. In any event, these findings are similar to those reported previously in Graves’ disease and Hashimoto’s thyroiditis (20). The frequency of fibrinogen deposition in Graves’ disease, however, was extremely low in comparison with that in Hashimoto’s thyroiditis. Werner et al. (30) and Kalderon and Bogaars (20) demonstrated no deposition of fibrinogen at the stroma and in the FBM in Graves’ disease. The underlying mechanism for such difference in ftbrinogen deposition between Graves’ disease and Hashimoto’s thyroiditis has to be clarified in future.
COMPLEMENT
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107
It is well known that the deposition of immunoglobulins and complement components in the glomerular basement membrane (GBM) shows a granular staining pattern which is regarded as the evidence for soluble immune complexes, e.g., DNA-anti-DNA immune complex in lupus nephritis (31-33). These data together with those reported in this paper indicate that the complement system may be locally activated in the thyroid glands with Graves’ disease via the classical pathway and that the deposition of soluble immune complexes may be present in the FBM of thyroids with Graves’ disease in a similar manner as found in the GBM of lupus nephritis. In summary the present study has provided us with evidence indicating that the complement system plays an important role in the pathogenesis of Graves’ disease and extensive study in this line is warranted. ACKNOWLEDGMENTS This work was supported in part by a research grant from the Ministry of Education, Science and Culture, Japan. We wish to thank Dr. T. Yoshida (Department of Pathology, University of Connecticut) and Dr. T. Miyahara (Division of Clinical Immunology, First Department of Surgery, Kyushu University) for their many helpful suggestions in this study. We express our thanks to Dr. S. Noguchi (Noguchi Hospital), Dr. A. Shiroozu and Dr. K. Inoue (Second Department of Medicine, Kyushu University) and Dr. F. Nakayama (First Department of Surgery, Kyushu University) for allowing US to study their patients. We also thank Mrs. K. Tasaki and Misses K. Matsui and M. Hiraki for their skillful technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. 15. 16. 17. 18. 19.
Adams, D. D., and Purves, H. D., Proc. Univ. Otago Med. Sch. 34, 11, 1956. Kriss, J. P., Pleshakov, V., and Chien, J. R., J. Clin. Endocrinol. 24, 1005, 1964. Miyai, K., and Werner, S. C., J. Clin. Endocrinol. 26, 504, 1966. Werner, S. C., and Tomasi, T. B., Jr., In “The Thyroid” (S. C. Werner and S. H. Ingbar, Eds.), 4th ed., pp. 606-623. Harper & Row, New York, 1978. Volpe, R., Farid, N. R., Von Westarp, C., and Row, V. V., Clin. Endocrinol. 3, 239, 1974. Holman, H. R., In “Immunological Diseases” (M. Samter, Ed.), 2nd ed., pp. 995- 1013. Little. Brown, Boston, 1971. Christian, C. L., In “Immunological Diseases” (M. Samter, Ed.), 2nd ed., pp. 1014- 1028. Little, Brown, Boston, 1971. Wilson, C. B., and Dixon, F. J., Kidney Znr. 5, 389, 1974. Stroud, M. B., Milir. Med. 135, 1105, 1970. Richman, R. A., Urmson, J. R., Dickson, S. L., Farnett, M. L., Stitzel, A. E., and Spitzer, R. E., Clin. Immunol. Immunopathol. 15, 600, 1980. Mayer, M. M., In “Experimental Immunochemistry” (E. A. Kabat and M. M. Mayer, Eds.), 2nd ed., pp. 135-139. Thomas, Springfield, Ill., 1961. Nishioka, K., J. Immunol. 90, 86, 1963. Nelson, R. A., Jr., Jensen, J., Gigli, I., and Tamura, N., Immunochemistry 3, 111, 1966. Fujiwara, H., Baba, T., Koitabashi, Y., Tasaki, K., Sunagawa, Y., Yasuda, K., and Esaki, H., “Serum Complement Activity in Thyroid Diseases.” Paper presented at the thirteenth Complement Symposium, Tokyo, Japan, September 1976. Ruddy, S., Everson, L. K., Schur, P. H., and Austen, K. F., J. Exp. Med. 134, (Suppl.), 259s. 1971. Hanauer, L. B., and Christian, C. L., Amer. J. Med. 42, 882, 1967. Nilsson, U. R., and Miiller-Eberhard, H. J., J. Exp. Med. 122, 277, 1965. Mancini, G., Carbonara. A. O., and Heremans, J. F., Immunochemistry 2, 235, 1965. Zollinger, H. U., and Mihatsch, M. J., In “Renal Pathology in Biopsy,” pp. 14-20. SpringerVerlag, Berlin, 1978.
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20. Kalderon, A. E., and Bogaars, H. A., Amer. J. Med. 63, 729, 1977. 21. Nastuk, W. L., Kessler, H. J., Grynbaum, A., Smith, M., and Herrmann, C., Arch. Neural. 15, 1, 1966. 22. Fukawa, M., Torisu, M., Miyahara, T., Harasaki, H., Kai, S., Yamamoto, H., Konomi, K., Nishimura, M., and Tanaka, J., Srtrgery 83, 293, 1978. 23. Kohler, P. F., and Ten Bensel, R., Clin. Exp. Immunol. 4, 191, 1969. 24. Calder, E. A., Penhale, W. J., Barnes, E. W., and Irvine, W. J.. &it. Med. .I. 2, 30, 1974. 25. Takeda, Y., and Kriss. J. P., J. Clin. Endocrinol. Metab. 44, 46, 1977. 26. Brohee, D., Delespesse, G., Debisschop, M. J., and Bonnyns, M., Clin. E.T~. Immunol. 36, 379, 1979. 27. Sonozaki, H., and Torisu, M., Ann. Rheum. Dis. 29, 164, 1970. 28. Rodman, W. S., Williams, R. C., Jr., Bilka, P. J., and Mtiller-Eberhard, H. J., J. Lab. Clin. Med. 69, 141, 1967. 29. Doniach, D., J. Clin. Pathol. 20, 385, 1967. 30. Werner, S. C., Wegelius, O., Fierer, J. A., and Hsu, K. C., N. Engl. J. Med. 287, 421, 1972. 31. Krishnan, C., and Kaplan, M. H., J. Clin. Invest. 46, 569, 1967. 32. Koffler, D., Agnello, V., Thoburn, R., and Kunkel, H. G.. 1. Exp. Med. 134 (Suppl.), 169s. 1971. 33. Koffler, D., Agnello, V., and Kunkel, H. G., Amer. J. Path. 74, 109, 1973.
IMMUNOLOGY
Immune
AND
IMMUNOPATHOLOGY
Complex
I. Complement
19, 98- 108 (1981)
Deposits in Thyroid Glands of Patients with Graves’ Disease:
System
in Serum and Thyroid Graves’ Disease
Gland of Patients
HIROSHI FUJIWARA, MOTOMICHI TORISU,’ TORU BABA,~ Division
of Clinical
immunology,
YASUSH~ AND HARUO ESAKI~
Department of First Surgery, Medicine, Fukuoka 812, Japan
Kyushu
with
KOITABASHI,* University
School
of
Received August 25, 1980 Serum complement level in 77 patients with Graves’ disease was periodically measured before and after various treatments including antithyroid drug administration and surgical operation. The complement titers showed remarkable fluctuation during the preoperative periods. However, the average complement titers in patients with this disease were not significantly different from those in patients with other thyroid diseases and in healthy volunteers. In patients whose clinical signs improved after surgical operation, the fluctuation of complement titers became smaller, falling within a normal range. To examine the possible local activation of complement system in thyroid glands of Graves’ disease, immunofluorescent stainings were performed in 30 thyroid glands with this disease utilizing fluorescein isothiocyanate (FITC)-labeled antisera against immunoglobulins and complement components. Granular deposition of IgG, IgA, IgB, Clq, C3, and C9 was observed along the follicular basement membrane (FBM) and/or at the stroma. IgG deposits along the FBM were usually accompanied with Clq, C3, and C9 deposition. None of 12 IgG-positive glands were stained by FITC-labeled antiserum to fibrinogen. Seven normal glands showed no significant staining for any of those immunoglobulins and complement components. These observations indicate that complement system may be locally activated in the thyroid gland via the classical pathway and that deposition of soluble immune complexes may occur in the FBM of Graves’ disease. Such local activation of complement may be in part the cause of strong fluctuation of complement levels in hyperthyroidism patients.
INTRODUCTION Antithyroid antibodies have been detected in patients with Graves’ disease as frequently as in those with Hashimoto’s thyroiditis. In addition, the discovery of long-acting thyroid stimulator (LATS) in Graves’ disease (1) and the subsequent demonstration that LATS is biologically and immunologically identical with im’ Address reprint requests to Motomichi Torisu, M.D., Division of Clinical Immunology, Department of First Surgery, Kyushu University School of Medicine, 3-l-l Maedashi Higashi-Ku, Fukuoka 812, Japan. * Present address: Department of Pediatrics, St. Marianna University School of Medicine, Kawasaki 213, Japan. 3 Present address: Department of Dermatology, Institute of Clinical Medicine, The University of Tsukuba, Ibaraki 300-3 1, Japan. * Present address: Department of Second Surgery, Hiroshima University School of Medicine, Hiroshima 734, Japan. 98 0090-1229/81/040098-11$01.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
COMPLEMENT
AND
GRAVES’
DISEASE
99
munoglobulin G (2, 3), prompted many investigators to perform various immunological studies on this disease (4, 5). However, its etiological details still remain unclear. The role of serum complement in the pathogenesis of systemic lupus erythematosus (SLE) (6), rheumatoid arthritis (RA) (7), and glomerulonephritis (8) was well documented. In contrast, serum complement studies in Graves’ disease and Hashimoto’s thyroiditis have been rather scarce (9, 10). In this communication, we will describe the interesting fluctuation of complement levels in patients with Graves’ disease and the evidence indicating the activation of complement system in thyroid glands of Graves’ disease. These results suggest the important role played by the complement system for the pathogenesis of Graves’ disease. MATERIALS
AND METHODS
Patients. This study was composed of 77 patients with Graves’ disease (15 males, 62 females; mean age of 34 + 12 years). These patients were confirmed by both clinical and laboratory findings, and classified.as follows according to their clinical status: (i) 12 untreated patients with hyperthyroid signs; (ii) 7 patients with the recurrent disease. They had undergone antithyroid drug therapy and/or operation. They had shown remarkable improvements clinically for several years after these treatments. However, when they visited our clinic again, they were found to have goiters and hyperthyroid symptoms; (iii) 58 patients under the treatment with antithyroid drugs (56 patients) or with the radioiodine therapy (2 patients). Two patients with radioiodine therapy had hyperthyroid signs during the current experimental period. As a control group, 57 patients with Hashimoto’s thyroiditis, 15 with welldifferentiated adenocarcinoma of the thyroid, 13 with thyroid adenoma, and 10 with adenomatous goiter were studied together with 500 healthy volunteers. Serum sampling. Approximately 10 ml of blood were obtained aseptically by venipuncture. After the blood was clotted at room temperature for approximately 1 hr, serum was separated by centrifugation at SOOgfor 10 min and stored at - 70°C until just prior to use. Serum thyroid hormone levels. Concentration of serum total thyroxin (T,) was measured by competitive protein-binding analysis using Res-O-Mat T, (Daiichi Radioisotope Laboratories, Ltd., Tokyo) (normal range, 5.0-13.7 p&/100 ml). Serum triiodothyronine (T,) was assayed by radioimmunoassay using RIA-Mat T, (Daiichi Radioisotope Laboratories, Ltd., Tokyo) (normal range, 90-210 ng/ 100 ml). Serum antithyroid antibodies. Thyroid antibodies to thyroglobulin (TGHA) and microsomes (MCHA) were measured by the tanned sheep red cell hemagglutination technique using thyroid test kit and microsome test kit, respectively (Fujizoki Pharmaceutical Co. Ltd., Tokyo). Serum complement assays. Total complement activity was measured in 50% hemolytic units (CH50) according to Mayer (11) and with the immune adherence hemagglutination (IA50) method of Nishioka (12). The activity of Cl, C4, and C2 was assayed hemolytically according to a modified method of Nelson et al. (13)
100
FUJIWARA
ET AL.
using microtiter plastic plates. The C3-C9 (C3T) activity was measured hemolytically as previously described (14). The C9 activity was titrated by immune hemolysis according to the method of Ruddy et al. (15). Zmmunochemicaf assay. Serum concentration of Clq subunit of Cl (16) and C5 (17) was determined as previously described using the single radial immunodiffusion method of Mancini et al. (18). The amount of C4 and C3 was measured using Partigen plates from Behring Institute, Marburg, West Germany. ZmmunojZuorescent studies. Thyroid tissues obtained at the time of operation or biopsy were quickly frozen in liquid nitrogen and stored at -70°C until examined. Tissue blocks were cut at 4-5 pm on a cryostat at -20°C. Fixation and immunofluorescent staining of tissue sections were performed according to the standard procedures (19). Presence of IgM, IgG, IgA, C3, and fibrinogen was demonstrated by direct immunofluorescent technique using FITC-labeled rabbit antisera (Behring Institute, Marburg, West Germany) to human IgM, IgG, IgA, C3, and fibrinogen, respectively. The demonstration of the presence of IgE, Clq, C4, C9, and C3 activator (C3A) was carried out by indirect immunofluorescent technique employing rabbit antisera to human IgE, Clq, C4, C9, and C3A (Behring Institute, Marburg, West Germany), respectively, and FITC-labeled goat antiserum to rabbit IgG (Melory Laboratories, Inc., Springfield, Va.). Statistical analysis. Experimental values in each group were analyzed by F test and Student’s t test. RESULTS Serum Complement
Activities
in Patients
with Graves’ Disease
The mean complement titers measured in patients with various thyroid diseases are shown in Table 1. The mean complement activity in Graves’ disease was 35.7 ? 8.1 CHSO/ml. No significant difference between this value and that of control patients or that of healthy volunteers was observed. However, the value of variance of the complement titers in patients with Graves’ disease and Hashimoto’s thyroiditis was significantly larger (P < 0.01) than that of healthy volunteers. The complement titers in 21 patients with Graves’ disease were periodically TABLE TOTAL COMPLEMENT ACTIVITY OF THE DISEASE, HASHIMOTO’S THYROIDITIS, No.
Diagnosis Graves’ disease Hashimoto’s thyroiditis Well-differentiated adenocarcinoma Adenoma Adenomatous goiter Healthy volunteers
1 SERA FROM PATIENTS AND OTHER THYROID
of
patients
WITH GRAVES’ DISEASES
Total complement activity (CHSO/ml; mean it 1 SD)
77 57 I5
35.7 i: 8.1 37.1 + 7.6 33.8 ” 4.4
13 10 500
36.5 +- 5.9 35.1 + 5.2 36.5 -c 5.8
COMPLEMENT
AND
GRAVES’
101
DISEASE
c9 unitlml
,o(
I Thyroid test 1#*~Microsome
3.9%
-8
4.1%
-6
-4
-2
0
2
4
test
6.7%
6
8 Time
10 12 24 36 in months
FIG. 1. Hemolytic complement titers, antithyroid antibody titers, and thyroid function tests in a patient with Graves’ disease before and after operation. Operation was performed at time zero. Hemolytic activities, especially C4 activities, were unstable during the preoperative course.
measured before and after treatments. Figures 1 and 2 show two typical cases of Graves’ disease with their various laboratory findings including complement and antithyroid antibody levels frequently measured before and after surgical operation. The changes of complement levels in.these cases were remarkably unstable during the preoperative period when serum thyroid hormone levels were elevated. However, after surgical operation, the fluctuation of complement levels became smaller, falling within a normal range. Thirteen out of twenty-one patients with this disease (62%) showed a similar fluctuation of complement levels as seen in these two cases during the preoperative period. However, the frequency of the preoperative fluctuation was not significantly different from that in healthy vol-
102
FUJIWARA
-6
-6
-4
-2
0
ET AL.
2
4
6
6 lime
10 12 24 36 in months
FIG. 2. Complement levels, antithyroid antibody titers, and thyroid function tests in a patient with Graves’ disease before and after operation. Operation was performed at time zero. Complement levels were unstable throughout the preoperative course. However, the levels became stable within normal limits after operation.
unteers. In alI 15 patients whose clinical signs improved after surgical operation, the complement titers became stable and maintained a normal level after operation. In Hashimoto’s thyroiditis, the complement levels in 6 out of 14 patients (43%) who were serially measured before and after treatment with thyroid hormone drugs showed a similar fluctuation as seen in Graves’ disease during the current experimental period. In contrast, no fluctuation of complement titers in patients with well-differentiated adenocarcinoma, adenoma, or adenomatous goiter was noticed.
COMPLEMENT
Relationship between Thyroid Graves’ Disease
AND
Hormone
GRAVES’
103
DISEASE
Levels and Complement
Activity
in
In order to explore a possible relationship between the degree of thyroid function and the complement activity in Graves’ disease, 77 patients with this disease were divided into three groups according to serum T4 level (see Materials and Methods). The mean complement titers in 24 patients with an elevated level of serum Tq, 37 patients with a normal level, and 16 patients with a low level were 37.9 + 9.2,35.7 t 7.6, and 36.3 k 7.8 CHSO/ml, respectively. No clear association existed between the thyroid hormone levels and the mean or variance of the complement titers in these groups. However, the value of standard deviation of the complement titers had a tendency to narrow with the improvement of clinical signs after appropriate treatments. Relationship between Thyroid Graves’ Disease
Antibodies
and Complement
Activity
in
The sera from 72 out of 77 patients with this disease (93%) contained thyroid antibodies (TGHA and/or MCHA) and no antibody was detected in the other five patients. The mean complement titer in patients with serum antithyroid antibodies was 36.0 2 8.0 CHSO/ml and that in the antibody-negative patients was 33.1 + 5.1 CHSO/ml. The difference of complement titers between these two groups was not statistically significant. Immunojluorescent Graves’ Disease
Studies on the Thyroid
Glands in Patients
with
The abnormal fluctuation of serum complement levels in patients with Graves’ disease as described above seems to suggest that the complement system may be involved in pathological process of this disease. Therefore, we have next explored for possible deposition of immunoglobulins and complement components in thyroid glands of Graves’ disease patients, utilizing both direct and indirect immunofluorescent staining techniques. As summarized in Table 2, granular deposition of immunoglobulins and complement components was recognized along the FBM in a significant number of glands examined; IgG deposition was found in 12 specimens (40%) out of 30 examined. Similarly detected were IgA (3%), IgE (17%), Clq (50%), C3 (20%), and C9 (88%). Immunofluorescent staining with C9 in this disease was performed only in IgG-positive thyroid specimens. In addition to the deposition along the FBM, immunoglobulins and complement components were also stained at the stroma of thyroid glands. Deposition of IgG, IgA, IgE, Clq, and C9 was detected in 27, 13, 22,58, and 13%, respectively, of the specimens examined. As also shown in Table 2, we could detect fibrinogen in 7 cases out of 24 thyroid glands examined. Interestingly, only one out of these seven specimens showed the concurrent deposition of IgE and Clq. The other six glands did not show any positive staining for immunoglobulins or complement components. Typical granular staining of IgG and C3 is demonstrated in Figs. 3 and 4, respectively. Staining pattern of IgA, IgE, Clq, C9, and fibrinogen was similar to that pictured in these figures.
104
FUJIWARA TABLE IMMUNOFLUORESCENT
STAINING
GRAVES’
DISEASE
Graves’ disease Expt
No.
Stroma
kM
30 30 30 23 12 7 30 8” 8 24
0 8 4 5 7 0 0 1 0 6
I@ Id I@ Ch c4 c3 c9 C3A Fib’
AND
ET AL. 2 OF THE
HASHIMOTO’S
THYROID
GLANDS
IN
THYROIDITIS
Hashimoto’s thyroiditis
Normal glands”
FBMb
No.
Stroma
FBM
No.
Stroma
FBM
0 12 1 4 6 0 6 7 0 1
17 17 17 17 14 10 17 9 10 12
6 14 10 8 3 2 2 3 2 10
0 3 2 2 I 1 2 2 1 0
7 7 7 1 7 NT” 7 NT NT 4
0 0 0 0 0 NT 0 NT NT 1
0 0 0 0 0 NT 0 NT NT 0
” Normal glands were obtained from the uninvolved tissue of nontoxic nodular goiter. ’ FBM: Follicular basement membrane. c Fib: Fibrinogen. ’ Immunofluorescent staining with C9 in Graves’ disease was performed only for IgG positive cases along the FBM. ( NT: Not tested
In order to compare these pathological findings with those in the well-known autoimmune thyroiditis, we have next examined thyroid glands with Hashimoto’s thyroiditis for staining of immunoglobulins and complement components. As expected, a significant number of specimens from this type of thyroiditis have shown positive staining for various immunoglobulins and complement components both along the FBM and in the stroma, in the same pattern as that of Graves’ disease. The stromal deposition of immunoglobulins and complement components except Clq was encountered more frequently in this disease than in Graves’ disease. In addition, stromal deposition of fibrinogen was recognized frequently in this disease (83%), whereas no FBM deposition of fibrinogen was found. These results are also summarized in Table 2. In contrast, none of the seven normal thyroid glands obtained from patients with nontoxic nodular goiter showed any specific staining for any types of immunoglobulins or complement components tested. These results provide stronger evidence that the complement system may have an important role in the pathological development of Graves’ disease as has been suggested by Kalderon and Bogaars for Graves’ disease and Hashimoto’s thyroiditis (20). DISCUSSION In the present communication, we have described that the serum complement activity in patients with Graves’ disease fluctuated conspicuously as well as in Hashimoto’s thyroiditis although the mean titer of the activity was not significantly different from that in other thyroid disease patients or healthy volunteers. The inability of Graves’ disease patients to maintain the complement activity at a stable level was corrected after surgical operation. These initial observations have led us to believe in the active participation of the complement system in this
COMPLEMENT
AND
GRAVES’
DISEASE
105
FIG. 3. Immunofluorescence demonstration of granular deposits of IgG along the FBM in a patient with Graves’ disease. This specimen showed positive staining for IgG, IgE, Clq, C3, and C9 along the FBM and at the stroma. Original magnification, x 100.
deposits are evident along the FBM. Original magnification,
106
FUJIWARA
ET AL.
disease process. We were able to show that various types of immunoglobulins (IgG, IgA, and IgE) and complement components (Clq, C3, and C9) were deposited along the FBM and in the stroma of thyroid glands. As far as we know, this is the first demonstration of IgG, Clq, C3, and C9 being found simultaneously deposited along the FBM in Graves’ disease. Such fluctuation of complement titers as seen in Graves’ disease has been repotted in myasthenia gravis (21, 22), which thymoma or thymic hyperplasia is frequently implicated. In addition, the fluctuation of the titers in myasthenia gravis became much smaller after surgical operation and, in some cases, fell within a normal range. However, its precise mechanism still remains unclear. The decrease in the whole complement activity and early components of complement (Clq, C4, C2, and C3) have been reported in SLE, a representative autoimmune disease (23). The mechanism of this depression is presumably due to activation and subsequent consumption of the complement system by the presence of circulating immune complexes. Although circulating immune complexes have been reported in Graves’ disease (24-26), the present study has revealed that serum complement titers did not show significant changes regardless of the presence or absence of circulating thyroid antibodies. These results suggest that the complement consumption may not occur in the circulation to such an extent that serum complement titers are significantly decreased in Graves’ disease. It is obvious, however, that further investigations are required for clarification of the underlying mechanism to explain these apparently conflicting observations. On the other hand, in RA, the level of serum complement activity is normal or even elevated, while the complement titer in synovial fluids is strikingly diminished in comparison with other joint diseases (27) and immunoglobulins and complement components are found deposited in the synovial membranes (28). These findings in RA seem to indicate the importance of local activation and subsequent consumption of the complement system without much effect on the serum complement level during a certain disease process. Actually in the present study we have found the granular deposition of IgG, Clq, C3, and C9 along the FBM area and in the stroma of thyroid glands with Graves’ disease, in spite of no apparent depression of the serum complement level. In contrast, the seven normal glands obtained from patients with nontoxic nodular goiter showed no significant staining along the FBM or in the stroma for immunoglobulins and complement components tested. In the present study, IgG deposition along the FBM was usually accompanied with Clq, C3, and C9 deposition. Although deposits of immunoglobulins and C3 component in the FBM of Graves’ thyroid glands were previously reported (29, 30), this is the first demonstration that IgG, Clq, C3, and C9 were deposited together along the FBM in this disease. In any event, these findings are similar to those reported previously in Graves’ disease and Hashimoto’s thyroiditis (20). The frequency of fibrinogen deposition in Graves’ disease, however, was extremely low in comparison with that in Hashimoto’s thyroiditis. Werner et al. (30) and Kalderon and Bogaars (20) demonstrated no deposition of fibrinogen at the stroma and in the FBM in Graves’ disease. The underlying mechanism for such difference in ftbrinogen deposition between Graves’ disease and Hashimoto’s thyroiditis has to be clarified in future.
COMPLEMENT
AND
GRAVES’
DISEASE
107
It is well known that the deposition of immunoglobulins and complement components in the glomerular basement membrane (GBM) shows a granular staining pattern which is regarded as the evidence for soluble immune complexes, e.g., DNA-anti-DNA immune complex in lupus nephritis (31-33). These data together with those reported in this paper indicate that the complement system may be locally activated in the thyroid glands with Graves’ disease via the classical pathway and that the deposition of soluble immune complexes may be present in the FBM of thyroids with Graves’ disease in a similar manner as found in the GBM of lupus nephritis. In summary the present study has provided us with evidence indicating that the complement system plays an important role in the pathogenesis of Graves’ disease and extensive study in this line is warranted. ACKNOWLEDGMENTS This work was supported in part by a research grant from the Ministry of Education, Science and Culture, Japan. We wish to thank Dr. T. Yoshida (Department of Pathology, University of Connecticut) and Dr. T. Miyahara (Division of Clinical Immunology, First Department of Surgery, Kyushu University) for their many helpful suggestions in this study. We express our thanks to Dr. S. Noguchi (Noguchi Hospital), Dr. A. Shiroozu and Dr. K. Inoue (Second Department of Medicine, Kyushu University) and Dr. F. Nakayama (First Department of Surgery, Kyushu University) for allowing US to study their patients. We also thank Mrs. K. Tasaki and Misses K. Matsui and M. Hiraki for their skillful technical assistance.
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