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Ultrastructural alterations of the follicular basement membrane in hashimoto's thyroiditis
Ultrastructural Alterations of the Follicular Basement Membrane in Hashimoto’s Thyroiditis Report of Eight Cases with Basement Membrane Deposits
ALBERT
E. KALDERON,
HENDRIK ISRAEL
A. BOGAARS, DIAMOND,
Providence.
M.D. M.D
M.D.
Rhode Island
From the Department of Pathology, Roger Hospital, Providence, Williams General Rhode Island and Section of Pathology, Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island. A part of this investigation was supported by the Brown-Hazen Fund. Requests for reprints should be addressed to Dr. Albert E. Kalderon, Roger Williams General Hospital, 825 Providence, Rhode Chalkstone Avenue, Island 02908. Manuscript accepted March 28, 1973.
Thyroid glands in eight cases, diagnosed histologically as typical of Hashimoto’s thyroiditis, were studied by electron microscopy with particular emphasis on the follicular basement membrane. Findings were compared with those in three cases of Graves’ disease (one associated with lymphocytic thyroiditis), one case of chronic lymphocytic thyroiditis and one case in which the thyroid was normal. In all cases of Hashimoto’s thyroiditis, distinct electron dense deposits were observed in the follicular basement membrane of some follicles, almost always associated with plasma cells in close apposition to the follicular basement membrane. In most instances, the electron dense deposits resembled the antigen-antibody deposits of immune complex nephropathies. Positive correlation with immunofluorescent studies was obtained in only one case of Hashimoto’s thyroiditis which was clinically associated with systemic lupus erythematosus. Positive immunofluorescent staining was also seen in the control cases of Graves’ disease but was not observed in the case of chronic lymphocytic thyroiditis and in the normal thyroid. Despite this apparent discrepancy, morphologic studies suggest that follicular basement membrane alterations in Hashimoto’s thyroiditis are a specific pathologic feature of the disease. Hashimoto [l], in 1912, described a new inflammatory disease of the thyroid gland under the name of Struma lymphomatosa. The morphologic changes of Hashimoto’s thyroiditis are characteristic of the disease and include infiltration of the thyroid tissue by lymphocytes and plasma cells, irregular fibrous replacement of the parenchyma, lymphoid nodules with germinative centers and focal destruction of follicles with oncocytic degeneration of follicular cells. Since the work of Rose and Witebsky [2], Hashimoto’s thyroiditis has been recognized as an autoimmune disorder in a large number of studies [3-51. Currently, two lines of thought dominate the pathogenesis of the disease: the theory of delayed hypersensitivity reaction [6] (cell-mediated immunity) and the “secluded antigen theory”
October
1973
The American
Journal of Medicine
Volume 55
485
BASEMENT
MEMBRANE
CHANGES
IN HASHIMOTO’S
THYROIDITIS-KALDERON
[7], the latter has been partly discredited since the discovery of circulating thyroglobulin antibodies in normal subjects. In the latter case, the disease was thought to result from a breakdown of the morphologic integrity of the thyroid follicle which presumably provides an immunologic basis for antibody formation against leaking thyroglobulin. Accordingly, thyroglobulin is considered to be a segregated protein that is shielded from the immunologic system by the tight junctions of the follicle cells and by the follicular basement membrane. Acceptance of the “leak hypothesis” by some has prompted several investigators to study the follicular basement membrane in various diseases associated with high thyroglobulin antibody titers. In this context, Sommers and Meissner [8] first described the discontinuities and degenerative changes of the follicular basement membrane in Hashimoto’s thyroiditis as discerned by light microscopy, and subsequently Stuart and Allan [9] confirmed the follicular basement membrane alterations and pointed to a direct relationship between basement membrane changes and high antibody titers. Among the few electron microscopic studies [lo-131 of Hashimoto’s thyroiditis, only Irvine and Muir [l l] mention occasional temporary gaps allowing the passage of inflammatory cells through an otherwise continuous membrane; in the other studies, no particular attention was given to the follicular basement membrane. This report deals with the ultrastructural alterations observed in the follicular basement membrane of eight cases of Hashimoto’s thyroiditis not previously recorded. These changes suggest that foliicular basement membrane alteration is an important pathologic feature of Hashimoto’s thyroiditis. MATERIALS
AND METHODS
Case Selection. The material we present is derived from a larger research project whereby all thyroid glands removed at surgery at Roger Williams General Hospital are systematically examined by electron microscopy and by histochemistry. The chance discovery of significant follicular basement membrane changes in one case of Hashimoto’s thyroiditis prompted reevaluation of all cases on file. This report is based on eight cases studied by electron microscopy during a 2 year period. In all cases selected, the diagnosis was made on morphologic grounds and correlated with the clinical diagnosis. Ultrastructural findings were compared with those in normal human thyroid tissue and in three cases of Graves’ disease; one associated with nonspecific chronic lymphocytic thyroiditis, and one. case of chronic lymphocytic thyroiditis.
486
October
1973
The American Journal of Medicine
ET AL.
Small slices of tissue samples Electron Microscopy. measuring approximately 2 mm in thickness were cut in small cubes and fixed in cold 2.5 per cent glutaraldehyde prepared in O.lM cacodylate buffer (pH 7.4) for 4 hours, postfixed with 1 per cent osmium tetroxide for 1 hour and embedded in Epon 812 [14]. Sections were cut with an LKB Ultratome III Ultra microtome. Thick sections (1 p) were stained with toluidine blue for light and electron microscopic correlation. Thin sections were supported on 200 mesh grids stained with uranyl acetate and lead citrate [15] and examined with an RCA EMU 4E electron microscope at 75 KV. lmmunofluorescent Studies. For direct immunofluorescence, tissues obtained at surgery were quick frozen in liquid nitrogen and stored at -70°C until examined. Tissue blocks were cut at 5 p on an IEC cryostat at -20°C. Sections were thawed at room temperature and fixed for 10 minutes in acetone. After washing in three changes of phosphate buffered saline (O.OlM, pH 7.2), the sections were overlaid with fluoresceinconjugated, commercially obtained (Hyland Laboratories, Los Angeles, Calif. and Hoechst Pharmaceuticals, Somerville, N.J.), goat and rabbit antiserums against human immunoglobulins G (IgG), A (IgA), M (IgM), betalc/,a globulin, fibrinogen, albumin and thyroglobulin, and incubated at room temperature for 30 minutes. The antiserums were tested for specificity by immunoelectrophoresis against normal human serum. Immunologic sensitivity was determined according to the method of Beutner et al. [16]. Conjugates were initially used in working dilutions of 0.25 to 0.50 units/ ml; later, examinations were repeated using dilutions of 1 unit/ml. All antiserums were absorbed before use with mouse liver powder. After incubation, sections were washed in three changes of phosphate buffered saline for a total of 15 minutes and mounted in phosphate buffered saline buffered glycerol. Examinations were carried out immediately and after 18 hours of storage at 4’C. Washed-unstained, and unwashedunstained sections were used as controls. Blocking procedures were carried out by prior incubation of sections with unconjugated antiserums against human IgG, IgA and IgM followed by the conjugated antiserums. The mounted sections were examined with a Leitz microscope equipped with fluorescent attachment using a HBO 200 mercury vapor lamp, UGt exciter filter, k430 barrier filter and cardioid dark field condenser. RESULTS Electron Microscopy. Electron microscopic studies were particularly directed to the follicular basement membrane. The normal human thyroid follicles disclosed a follicular basement membrane which was discrete and homogeneous in electron density displaying a uniform thickness throughout (Figure 1). In all cases of Hashimoto’s thyroiditis, distinct alterations affecting the follicu-
Volume 55
Figure 1. Basal portion of a normal human thyroid folk- _ cle. Arrow points to a homo- “’ geneous discrete follicular basement membrane. Ca = capillary; Fc = follicular cell. Original magnification X 13,400. reduced by 15 per cent.
Ca ’
Basal portion of Figure 2. follicular cells in Hashimoto’s thyroiditis. Note uniform thickness and irregularity Of the basement membrane (FBM). Original magnification X 13,400, reduced by 15 per cent.
Figure 3. Basal portion of a follicular cell in Hashimoto’s thyroiditis. Note extensive lamellation and duplication of the follicular basement membrane. Co = perifollicular collagen. Original magnificatfon X 18,000, reduced by 15 per cent.
~~~~*~~,,~~~“‘~2 %$
i.6 ‘t;;‘-
BASEMENT
MEMBRANE
CHANGES
IN HASHIMOTO’S
THYROIDITIS-KALDERON
lar basement membrane were noted. These changes were almost entirely confined to the areas of heavy cellular infiltration of the thyroid parenchyma. In these areas, the follicular basement membrane invariably showed diffuse, irregular, thickening and blurring (Figure 2) which sometimes appeared fibrillar. Occasionally, reduplication assuming a multilayered aspect was ob-
ET AL
served (Figure 3). There was no interruption of the follicular basement membrane, and no persistent gaps were noted except for instances in which plasma cells or lymphocytes were in transit from the perifollicular spaces to the acini. Tendency of a basement membrane-like substance to pile up, penetrate and divide adjacent follicular seen. The most significant characteristic
cells was finding
Figure 4. The follicular basement membrane in Hashimoto’s thyroiditis. Note the irregular thickening with globoid electron dense material. Original magnification X 22,000, reduced by 15 per cent.
Figure 5. The follicular basement membrane in Hashimoto’s thyroiditis showing electron dense deposits (arrows). Note penetration of the basement membrane containing deposits between two neighboring follicular cells. Original magnification X 11,200, reduced by 10 per cent.
488
October 1973
The American Journal of Medicine
Volume 55
BASEMENT
was the presence of dense material sharply
irregular globoid contrasting with
MEMBRANE
electron the elec-
tron opacity of the surrounding follicular basement membrane (Figure 4). In the majority of the cases the deposits were seen in the follicular basement membrane where plasma cells closely abutted the follicle. In only one case were the deposits noticed in a portion of the thyroid that was not infiltrated by cells. Noninfiltrated areas showed no significant follicular basement membrane alterations and exhibited characteristics similar to the follicular basement membrane of normal thyroid. In general, the deposits were found with relative ease; however, in some instances it was necessary to study several additional blocks to demonstrate their presence. No deposits were found in any of the control cases even after a diligent search. In Hashimoto’s thyroiditis, areas with a predominantly lymphocytic infiltrate failed to reveal the follicular basement membrane deposits which appeared to be essentially associated with a plasmacytic cell population (Figure 5). In Graves’ disease and in chronic nonspecific lymphocytic thyroiditis, follicular basement membrane changes were inconspicuous and at times limited only to a minimal irregular thickening without the follicular basement membrane deposits observed in Hashimoto’s thyroiditis. Positive staining of follicuImmunofluorescence.
CHANGES
IgG and
IN HASHIMOTO’S
IgM.
THYROIDITIS
It was sometimes
KALDERON
difficult
ET
AL
to visual-
ize follicles amid the inflammatory exudate and scar tissue; however, in all cases, single or groups of follicles were found after adequate sampling of the specimens. Selection of blocks was aided by the positive staining with fluorescinated antithyroglobulin serum of the colloid material. In only one case of Hashimoto’s thyroiditis, clinically associated with systemic lupus erythematosus, was there heavy granular immunofluorescent staining of the follicular basement membrane with IgG, IgA and beta,a/,c globulin (Figure 6). A slight, nonspecific, fluorescence of follicular cell cytoplasm was irregularly present. The absence of follicular basement membrane staining in the majority of cases of thyroiditis was in contrast to the prominent granular staining of the follicular basement membrane with anti-IgG, IgM and beta,a/lc globulin in all cases of Graves’ disease examined (Figure 7). I mmunofluorescent staining was absent also in the control cases of chronic lymphocytic thyroiditis and in normal human thyroid tissue obtained during a parathyroidectomy procedure. There was no staining for fibrinogen. With antithyroglobulin serums, staining was limited to the follicular lumen with some occasional staining in the follicular cells and in the cytoplasm of the infiltrating cells. COMMENTS
lar basement membrane was not observed with any of the conjugated antiserums used. Varying degrees of staining of the stroma and of the cytoplasm of immunocompetent cells was obtained IgA and IgM, and complement with anti-IgG,
Electron microscopy performed in eight cases of Hashimoto’s thyroiditis unequivocally revealed a variety of alterations in the follicular basement membrane including the presence in all cases of
could be demonstrated in the stroma in cases. Faint staining of the follicular colloid observed occasionally after treatment with
electron dense deposits which were in control cases. In only one case posits positive on immunofluorescent
most was anti-
of- the same specimen pictured in Figure 5. Granular and short linear deposits are evident in the follicular basement membrane. Original magnification X 400, slightly reduced.
October
not observed were the destaining and,
Graves’ disease showing immunofluorescent staining with anti-IgG. Granular and short linear deposits are evident in the follicular basement membrane. Original magnification X 400. slightly magnified.
1973
The American Journal of Medicine
Volume 55
489
BASEMENT
MEMBRANE
CHANGES
IN HASHIMOTO’S
THYROIDITIS-KALDERON
therefore, strongly suggestive of immune complex deposits. Morphologically, the electron dense material in Hashimoto’s thyroiditis, resembles the electron dense material described in a variety of nephropathies [17] as immune complex deposits. Similarly, in Goodpasture’s syndrome, it has been shown that immune deposits are localized. in the basement membrane of the lung and kidney and there is evidence to implicate glomerular basement membrane antibodies as the source of morphologic damage [18]. We recently encountered a case of Goodpasture’s syndrome associated with Hashimoto’s thyroiditis indicating perhaps an autoimmune process not only involving the kidneys and lungs but also the thyroid. Basement membrane damage in Goodpasture’s syndrome has been documented in the form of irregular thickening and mottling [19]. In idiopathic pulmonary hemosiderosis [20], electron dense deposits were observed in the pulmonary capillary basement membrane structurally resembling electron dense deposits encountered in immune complex nephritis. Experimentally, Weigle and Nakamura [21] were able to produce complex-induced “chronic glomerulonephritis” following injections of altered thyroglobulin which resulted in the deposition of thyroglobulin-antithyroglobulin complexes and complement along the glomerular basement membrane while inducing autoimmune thyroiditis. However, they do not mention that the follicular basement membrane also acquired the same deposits that were seen in the glomerular basement membrane. Recently, Werner et al. [22] demonstrated, by immunofluorescence, the presence of immune deposits in the follicular basement membrane of a large group of cases of Graves’ disease. Our immunofluorescent findings of immune deposits in the follicular basement membrane of three control cases of Graves’ disease corroborates their observation. However, we were unable to demonstrate electron dense deposits in the follicular basement membrane in any of the cases of our control group, including the cases of Graves’ disease in which the follicular basement membrane showed positive staining. In an extensive ultrastructural study of multinodular goiter and Graves’ disease, Heimann [23] reported no significant changes in the follicular basement membrane other than irregular thickening. The inability to demonstrate immunoglobulins and complement in the follicular basement membrane in seven of eight cases of Hashimoto’s thyroiditis raises the question whether they are indeed the result of an immunologic event. Our conclusions are based on the morphologic similarity
490
October 1973
The American Journal of Medicine
ET AL
between the electron dense masses and the deposits in immune complex diseases and are strengthened by the demonstration of immunoglobulins and complement in one case. Failure to detect these proteins in the remaining seven cases may be the result of the relatively small number of deposits present, loss of antigenicity due to degradation of the immune complex components or lack of free antigen and antibody sites analogous with a similar situation in some of the cases of hereditary nephritis reported by Spear et al. [24]. The finding by Gerber and Paronetto [25] of IgE deposits in a granular and short linear manner in the glomeruli of patients with nephrotic syndrome and absence of other immunoglobulin and complement in these cases, also deserves attention in this regard. In our immunofluorescent studies, no anti-IgE was used. Previous electron microscopic studies of Hashimoto’s thyroiditis in man [10,11,13] as well as studies of experimentally-induced chronic thyroiditis [26] failed to yield information regarding specific changes involving the follicular basement membrane as reported in this study. The constant presence of electron dense deposits in Hashimoto’s thyroiditis as seen by electron microscopy appear to be significant and suggest perhaps the participation of the follicular basement membrane in the pathogenesis of the disease in a yet undetermined manner. It is certain, however, that the morphologic analogy concerning the follicular basement membrane alterations between Hashimoto’s thyroiditis and immune complex nephropathies cannot be extended to include etiologic mechanisms nor can it imply similar pathogenic factors without more substantial evidence. Electron dense deposits in the follicular basement membrane of patients with Hashimoto’s thyroiditis may perhaps point to the participation of the basement membrane of yet another antigenie factor in the disease, besides those already known, i.e., thyroglobulin, microsomal and second colloid antigens. The fact that the electron dense deposits were seen almost exclusively in areas of dense cellular infiltrate in close association with plasma cells provides circumstantial evidence that the deposits may be associated with immunoglobulins secreted locally by the plasma cells. Whatever the case may be, the pathogenic order in which follicular basement membrane alterations occur in the sequence of events in Hashimoto’s thyroiditis is not well understood. As pointed out by Hall [27], the possibility exists that follicular basement membrane alterations may be
Volume 55
BASEMENT
secondary Future
to a yet obscure
Investigations
should
role played by the follicular in autoimmune thyroiditis.
autoimmune consider basement
MEMBRANE
process.
CHANGES
IN HASHIMOTO’S
THYROIDITIS
KALDERON
ET Al-
ACKNOWLEDGMENT
the possible membrane
We wish to express our gratitude to Mr. Jolly, Mrs. Randi Shorthouse and to Mrs. Aleszczyk for their technical assistance.
Grant Lillian
REFERENCES
2.
3.
4.
5. 6. 7.
a.
9.
10.
11.
12.
13.
14. 15.
Hashimoto H: Zur Denntriss der lymphomatosen Veranderung der Schilddruse (Struma lymphomatosa). Langenbecks Arch Klin Chir 97: 219, 1912. Rose Nf?, Witebsky E: Studies on organ specificity. Changes in the thyroid glands of rabbits following active immunization with rabbit thyroid extracts. J lmmun 76: 417, 1958. McKay IR, Burnett FM: Auto-immune disease: experimental and clinical. Proc Roy Sot Med 55: 619, 1962. Doniach D. Riott IM: Autoimmune Thyroid Disease, Textbook of Immunopathology. vol 2 (Miescher AP, Muller-Eberhard HJ, eds). New York, Grune & Stratton, 1969. Donlach D: Thyroid autoimmune disease. J Clin Path 20: 385, 1967. Roitt IM, Doniach D: Delayed hypersensitivity in autoimmune disease. Brit Med Bull 23: 66, 1967. Roitt IM, Campbell PN. Doniach D: The nature of the gamma globulins in lymphadenoid goiter. Biochem J 69: 248, 1958. Sommers CS, Meissner WA: Basement membrane changes in chronic thyroiditis and other thyroid diseases. Amer J Clin Path 21: 434, 1954. Stuart AE, Allan WS: The significance of basement membrane changes in thyroid disease. The Lancet 2: 1204, 1958. N&e P: Ultrastructure of the thyroid in a case of Hashimoto’s goiter (struma lymphomatosa). Path Europ 1: 234, 1966. Irvine WJ. Muir AR: An electron microscopic study of Hashimoto’s thyroiditis. Quart J Exp Physiol 46: 13, 1963. N&e P: The ultrastructure of the thyroid in chronic autoimmume thyroiditis. Virchows Arch Path Anat 346: 302.1969. Lupulescu A, Petrovici A: Ultrastructure of the thyroid gland, Ultrastructure of the Thyroid in Lymphocytic Thyroiditis (Hashimoto’s Disease), Baltimore, Williams 8. Wilkins, 1966, p 74. Luft J: Improvement in epoxy embedding methods. J Cell Biol9: 409, 1961. Reynaulds ES: The use of the lead citrate at high pH as
16.
17.
ia.
19.
20.
21.
22.
23. 24.
25.
26.
27.
October 1973
electron opaque stain in electron microscopy J Cell Biol 17: 208, 1963. Beutner EH, Sepulveda MR. Barnett EV: Quantitative studies of immunofluorescent staining. Bull WHO 39: 587, 1968. Churg J: Electron microscopic aspects of renal pathology, Structural Basis of Renal Disease (Becker EL, ed), New York, Harper & Row, 1966, p 132. Lerner RA, Glassock RJ, Dixon FJ: The role of antiglomerular basement membrane antibody in the pathogenesis of human glomerulonephritis. J Exp Med 126: 989,1967. Batting AJ, Brown AL, Divertie MB: The pulmonary lesion in a patient with Goodpasture’s syndrome as studied with the electron microscope. Amer J Clin Path 42: 387, 1964. Elliott A, Kuhn C: Idiopathic pulmonary hemosiderosis. Ultrastructural abnormalities in capillary walls. Amer Rev Resp Dis 102: 695, 1970. Weigle WO, Nakamura RM: Perpetuation of autoimmune thyroiditis and production of secondary renal lesions following periodic injections of aqueous preparations of altered thyroglobulin. Clin Exp lmmun 4: 645, 1969. Werner SC, Wegelius 0, Fierer JA, Hsu KC: Immunoglobulins (E.M,G) and complement in connective tissues of the thyroid in Graves’ disease. New Eng J Med 267: 421, 1972. Heimann P: Ultrastructure of human thyroid. Acta Endoer 53 (suppl 110): 1, 1966. Spear GS, Whitworth JM, Komgsmark BW: Hereditary nephritis with nerve deafness: immunofluorescent studies on the kidney, with a consideration of discordant immunoglobulin-complement immunofluorescent reactions. Amer J Med 49: 52, 1970. Gerber MA, Paronetto F: IgE in glomeruli of patients with nephrotic syndrome. The Lancet I: 1097, 1971. Sobel H, Geller J: Experimental thyroiditis in guinea pig: electron microscopy. Amer J Path 46: 149, 1965. Hall R: Immunologic aspects of thyroid function. New Eng J Med 266: 1204,1962.
The American Journal of Medicine
Volume 55
491
ALBERT
E. KALDERON,
HENDRIK ISRAEL
A. BOGAARS, DIAMOND,
Providence.
M.D. M.D
M.D.
Rhode Island
From the Department of Pathology, Roger Hospital, Providence, Williams General Rhode Island and Section of Pathology, Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island. A part of this investigation was supported by the Brown-Hazen Fund. Requests for reprints should be addressed to Dr. Albert E. Kalderon, Roger Williams General Hospital, 825 Providence, Rhode Chalkstone Avenue, Island 02908. Manuscript accepted March 28, 1973.
Thyroid glands in eight cases, diagnosed histologically as typical of Hashimoto’s thyroiditis, were studied by electron microscopy with particular emphasis on the follicular basement membrane. Findings were compared with those in three cases of Graves’ disease (one associated with lymphocytic thyroiditis), one case of chronic lymphocytic thyroiditis and one case in which the thyroid was normal. In all cases of Hashimoto’s thyroiditis, distinct electron dense deposits were observed in the follicular basement membrane of some follicles, almost always associated with plasma cells in close apposition to the follicular basement membrane. In most instances, the electron dense deposits resembled the antigen-antibody deposits of immune complex nephropathies. Positive correlation with immunofluorescent studies was obtained in only one case of Hashimoto’s thyroiditis which was clinically associated with systemic lupus erythematosus. Positive immunofluorescent staining was also seen in the control cases of Graves’ disease but was not observed in the case of chronic lymphocytic thyroiditis and in the normal thyroid. Despite this apparent discrepancy, morphologic studies suggest that follicular basement membrane alterations in Hashimoto’s thyroiditis are a specific pathologic feature of the disease. Hashimoto [l], in 1912, described a new inflammatory disease of the thyroid gland under the name of Struma lymphomatosa. The morphologic changes of Hashimoto’s thyroiditis are characteristic of the disease and include infiltration of the thyroid tissue by lymphocytes and plasma cells, irregular fibrous replacement of the parenchyma, lymphoid nodules with germinative centers and focal destruction of follicles with oncocytic degeneration of follicular cells. Since the work of Rose and Witebsky [2], Hashimoto’s thyroiditis has been recognized as an autoimmune disorder in a large number of studies [3-51. Currently, two lines of thought dominate the pathogenesis of the disease: the theory of delayed hypersensitivity reaction [6] (cell-mediated immunity) and the “secluded antigen theory”
October
1973
The American
Journal of Medicine
Volume 55
485
BASEMENT
MEMBRANE
CHANGES
IN HASHIMOTO’S
THYROIDITIS-KALDERON
[7], the latter has been partly discredited since the discovery of circulating thyroglobulin antibodies in normal subjects. In the latter case, the disease was thought to result from a breakdown of the morphologic integrity of the thyroid follicle which presumably provides an immunologic basis for antibody formation against leaking thyroglobulin. Accordingly, thyroglobulin is considered to be a segregated protein that is shielded from the immunologic system by the tight junctions of the follicle cells and by the follicular basement membrane. Acceptance of the “leak hypothesis” by some has prompted several investigators to study the follicular basement membrane in various diseases associated with high thyroglobulin antibody titers. In this context, Sommers and Meissner [8] first described the discontinuities and degenerative changes of the follicular basement membrane in Hashimoto’s thyroiditis as discerned by light microscopy, and subsequently Stuart and Allan [9] confirmed the follicular basement membrane alterations and pointed to a direct relationship between basement membrane changes and high antibody titers. Among the few electron microscopic studies [lo-131 of Hashimoto’s thyroiditis, only Irvine and Muir [l l] mention occasional temporary gaps allowing the passage of inflammatory cells through an otherwise continuous membrane; in the other studies, no particular attention was given to the follicular basement membrane. This report deals with the ultrastructural alterations observed in the follicular basement membrane of eight cases of Hashimoto’s thyroiditis not previously recorded. These changes suggest that foliicular basement membrane alteration is an important pathologic feature of Hashimoto’s thyroiditis. MATERIALS
AND METHODS
Case Selection. The material we present is derived from a larger research project whereby all thyroid glands removed at surgery at Roger Williams General Hospital are systematically examined by electron microscopy and by histochemistry. The chance discovery of significant follicular basement membrane changes in one case of Hashimoto’s thyroiditis prompted reevaluation of all cases on file. This report is based on eight cases studied by electron microscopy during a 2 year period. In all cases selected, the diagnosis was made on morphologic grounds and correlated with the clinical diagnosis. Ultrastructural findings were compared with those in normal human thyroid tissue and in three cases of Graves’ disease; one associated with nonspecific chronic lymphocytic thyroiditis, and one. case of chronic lymphocytic thyroiditis.
486
October
1973
The American Journal of Medicine
ET AL.
Small slices of tissue samples Electron Microscopy. measuring approximately 2 mm in thickness were cut in small cubes and fixed in cold 2.5 per cent glutaraldehyde prepared in O.lM cacodylate buffer (pH 7.4) for 4 hours, postfixed with 1 per cent osmium tetroxide for 1 hour and embedded in Epon 812 [14]. Sections were cut with an LKB Ultratome III Ultra microtome. Thick sections (1 p) were stained with toluidine blue for light and electron microscopic correlation. Thin sections were supported on 200 mesh grids stained with uranyl acetate and lead citrate [15] and examined with an RCA EMU 4E electron microscope at 75 KV. lmmunofluorescent Studies. For direct immunofluorescence, tissues obtained at surgery were quick frozen in liquid nitrogen and stored at -70°C until examined. Tissue blocks were cut at 5 p on an IEC cryostat at -20°C. Sections were thawed at room temperature and fixed for 10 minutes in acetone. After washing in three changes of phosphate buffered saline (O.OlM, pH 7.2), the sections were overlaid with fluoresceinconjugated, commercially obtained (Hyland Laboratories, Los Angeles, Calif. and Hoechst Pharmaceuticals, Somerville, N.J.), goat and rabbit antiserums against human immunoglobulins G (IgG), A (IgA), M (IgM), betalc/,a globulin, fibrinogen, albumin and thyroglobulin, and incubated at room temperature for 30 minutes. The antiserums were tested for specificity by immunoelectrophoresis against normal human serum. Immunologic sensitivity was determined according to the method of Beutner et al. [16]. Conjugates were initially used in working dilutions of 0.25 to 0.50 units/ ml; later, examinations were repeated using dilutions of 1 unit/ml. All antiserums were absorbed before use with mouse liver powder. After incubation, sections were washed in three changes of phosphate buffered saline for a total of 15 minutes and mounted in phosphate buffered saline buffered glycerol. Examinations were carried out immediately and after 18 hours of storage at 4’C. Washed-unstained, and unwashedunstained sections were used as controls. Blocking procedures were carried out by prior incubation of sections with unconjugated antiserums against human IgG, IgA and IgM followed by the conjugated antiserums. The mounted sections were examined with a Leitz microscope equipped with fluorescent attachment using a HBO 200 mercury vapor lamp, UGt exciter filter, k430 barrier filter and cardioid dark field condenser. RESULTS Electron Microscopy. Electron microscopic studies were particularly directed to the follicular basement membrane. The normal human thyroid follicles disclosed a follicular basement membrane which was discrete and homogeneous in electron density displaying a uniform thickness throughout (Figure 1). In all cases of Hashimoto’s thyroiditis, distinct alterations affecting the follicu-
Volume 55
Figure 1. Basal portion of a normal human thyroid folk- _ cle. Arrow points to a homo- “’ geneous discrete follicular basement membrane. Ca = capillary; Fc = follicular cell. Original magnification X 13,400. reduced by 15 per cent.
Ca ’
Basal portion of Figure 2. follicular cells in Hashimoto’s thyroiditis. Note uniform thickness and irregularity Of the basement membrane (FBM). Original magnification X 13,400, reduced by 15 per cent.
Figure 3. Basal portion of a follicular cell in Hashimoto’s thyroiditis. Note extensive lamellation and duplication of the follicular basement membrane. Co = perifollicular collagen. Original magnificatfon X 18,000, reduced by 15 per cent.
~~~~*~~,,~~~“‘~2 %$
i.6 ‘t;;‘-
BASEMENT
MEMBRANE
CHANGES
IN HASHIMOTO’S
THYROIDITIS-KALDERON
lar basement membrane were noted. These changes were almost entirely confined to the areas of heavy cellular infiltration of the thyroid parenchyma. In these areas, the follicular basement membrane invariably showed diffuse, irregular, thickening and blurring (Figure 2) which sometimes appeared fibrillar. Occasionally, reduplication assuming a multilayered aspect was ob-
ET AL
served (Figure 3). There was no interruption of the follicular basement membrane, and no persistent gaps were noted except for instances in which plasma cells or lymphocytes were in transit from the perifollicular spaces to the acini. Tendency of a basement membrane-like substance to pile up, penetrate and divide adjacent follicular seen. The most significant characteristic
cells was finding
Figure 4. The follicular basement membrane in Hashimoto’s thyroiditis. Note the irregular thickening with globoid electron dense material. Original magnification X 22,000, reduced by 15 per cent.
Figure 5. The follicular basement membrane in Hashimoto’s thyroiditis showing electron dense deposits (arrows). Note penetration of the basement membrane containing deposits between two neighboring follicular cells. Original magnification X 11,200, reduced by 10 per cent.
488
October 1973
The American Journal of Medicine
Volume 55
BASEMENT
was the presence of dense material sharply
irregular globoid contrasting with
MEMBRANE
electron the elec-
tron opacity of the surrounding follicular basement membrane (Figure 4). In the majority of the cases the deposits were seen in the follicular basement membrane where plasma cells closely abutted the follicle. In only one case were the deposits noticed in a portion of the thyroid that was not infiltrated by cells. Noninfiltrated areas showed no significant follicular basement membrane alterations and exhibited characteristics similar to the follicular basement membrane of normal thyroid. In general, the deposits were found with relative ease; however, in some instances it was necessary to study several additional blocks to demonstrate their presence. No deposits were found in any of the control cases even after a diligent search. In Hashimoto’s thyroiditis, areas with a predominantly lymphocytic infiltrate failed to reveal the follicular basement membrane deposits which appeared to be essentially associated with a plasmacytic cell population (Figure 5). In Graves’ disease and in chronic nonspecific lymphocytic thyroiditis, follicular basement membrane changes were inconspicuous and at times limited only to a minimal irregular thickening without the follicular basement membrane deposits observed in Hashimoto’s thyroiditis. Positive staining of follicuImmunofluorescence.
CHANGES
IgG and
IN HASHIMOTO’S
IgM.
THYROIDITIS
It was sometimes
KALDERON
difficult
ET
AL
to visual-
ize follicles amid the inflammatory exudate and scar tissue; however, in all cases, single or groups of follicles were found after adequate sampling of the specimens. Selection of blocks was aided by the positive staining with fluorescinated antithyroglobulin serum of the colloid material. In only one case of Hashimoto’s thyroiditis, clinically associated with systemic lupus erythematosus, was there heavy granular immunofluorescent staining of the follicular basement membrane with IgG, IgA and beta,a/,c globulin (Figure 6). A slight, nonspecific, fluorescence of follicular cell cytoplasm was irregularly present. The absence of follicular basement membrane staining in the majority of cases of thyroiditis was in contrast to the prominent granular staining of the follicular basement membrane with anti-IgG, IgM and beta,a/lc globulin in all cases of Graves’ disease examined (Figure 7). I mmunofluorescent staining was absent also in the control cases of chronic lymphocytic thyroiditis and in normal human thyroid tissue obtained during a parathyroidectomy procedure. There was no staining for fibrinogen. With antithyroglobulin serums, staining was limited to the follicular lumen with some occasional staining in the follicular cells and in the cytoplasm of the infiltrating cells. COMMENTS
lar basement membrane was not observed with any of the conjugated antiserums used. Varying degrees of staining of the stroma and of the cytoplasm of immunocompetent cells was obtained IgA and IgM, and complement with anti-IgG,
Electron microscopy performed in eight cases of Hashimoto’s thyroiditis unequivocally revealed a variety of alterations in the follicular basement membrane including the presence in all cases of
could be demonstrated in the stroma in cases. Faint staining of the follicular colloid observed occasionally after treatment with
electron dense deposits which were in control cases. In only one case posits positive on immunofluorescent
most was anti-
of- the same specimen pictured in Figure 5. Granular and short linear deposits are evident in the follicular basement membrane. Original magnification X 400, slightly reduced.
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not observed were the destaining and,
Graves’ disease showing immunofluorescent staining with anti-IgG. Granular and short linear deposits are evident in the follicular basement membrane. Original magnification X 400. slightly magnified.
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therefore, strongly suggestive of immune complex deposits. Morphologically, the electron dense material in Hashimoto’s thyroiditis, resembles the electron dense material described in a variety of nephropathies [17] as immune complex deposits. Similarly, in Goodpasture’s syndrome, it has been shown that immune deposits are localized. in the basement membrane of the lung and kidney and there is evidence to implicate glomerular basement membrane antibodies as the source of morphologic damage [18]. We recently encountered a case of Goodpasture’s syndrome associated with Hashimoto’s thyroiditis indicating perhaps an autoimmune process not only involving the kidneys and lungs but also the thyroid. Basement membrane damage in Goodpasture’s syndrome has been documented in the form of irregular thickening and mottling [19]. In idiopathic pulmonary hemosiderosis [20], electron dense deposits were observed in the pulmonary capillary basement membrane structurally resembling electron dense deposits encountered in immune complex nephritis. Experimentally, Weigle and Nakamura [21] were able to produce complex-induced “chronic glomerulonephritis” following injections of altered thyroglobulin which resulted in the deposition of thyroglobulin-antithyroglobulin complexes and complement along the glomerular basement membrane while inducing autoimmune thyroiditis. However, they do not mention that the follicular basement membrane also acquired the same deposits that were seen in the glomerular basement membrane. Recently, Werner et al. [22] demonstrated, by immunofluorescence, the presence of immune deposits in the follicular basement membrane of a large group of cases of Graves’ disease. Our immunofluorescent findings of immune deposits in the follicular basement membrane of three control cases of Graves’ disease corroborates their observation. However, we were unable to demonstrate electron dense deposits in the follicular basement membrane in any of the cases of our control group, including the cases of Graves’ disease in which the follicular basement membrane showed positive staining. In an extensive ultrastructural study of multinodular goiter and Graves’ disease, Heimann [23] reported no significant changes in the follicular basement membrane other than irregular thickening. The inability to demonstrate immunoglobulins and complement in the follicular basement membrane in seven of eight cases of Hashimoto’s thyroiditis raises the question whether they are indeed the result of an immunologic event. Our conclusions are based on the morphologic similarity
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between the electron dense masses and the deposits in immune complex diseases and are strengthened by the demonstration of immunoglobulins and complement in one case. Failure to detect these proteins in the remaining seven cases may be the result of the relatively small number of deposits present, loss of antigenicity due to degradation of the immune complex components or lack of free antigen and antibody sites analogous with a similar situation in some of the cases of hereditary nephritis reported by Spear et al. [24]. The finding by Gerber and Paronetto [25] of IgE deposits in a granular and short linear manner in the glomeruli of patients with nephrotic syndrome and absence of other immunoglobulin and complement in these cases, also deserves attention in this regard. In our immunofluorescent studies, no anti-IgE was used. Previous electron microscopic studies of Hashimoto’s thyroiditis in man [10,11,13] as well as studies of experimentally-induced chronic thyroiditis [26] failed to yield information regarding specific changes involving the follicular basement membrane as reported in this study. The constant presence of electron dense deposits in Hashimoto’s thyroiditis as seen by electron microscopy appear to be significant and suggest perhaps the participation of the follicular basement membrane in the pathogenesis of the disease in a yet undetermined manner. It is certain, however, that the morphologic analogy concerning the follicular basement membrane alterations between Hashimoto’s thyroiditis and immune complex nephropathies cannot be extended to include etiologic mechanisms nor can it imply similar pathogenic factors without more substantial evidence. Electron dense deposits in the follicular basement membrane of patients with Hashimoto’s thyroiditis may perhaps point to the participation of the basement membrane of yet another antigenie factor in the disease, besides those already known, i.e., thyroglobulin, microsomal and second colloid antigens. The fact that the electron dense deposits were seen almost exclusively in areas of dense cellular infiltrate in close association with plasma cells provides circumstantial evidence that the deposits may be associated with immunoglobulins secreted locally by the plasma cells. Whatever the case may be, the pathogenic order in which follicular basement membrane alterations occur in the sequence of events in Hashimoto’s thyroiditis is not well understood. As pointed out by Hall [27], the possibility exists that follicular basement membrane alterations may be
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to a yet obscure
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role played by the follicular in autoimmune thyroiditis.
autoimmune consider basement
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ACKNOWLEDGMENT
the possible membrane
We wish to express our gratitude to Mr. Jolly, Mrs. Randi Shorthouse and to Mrs. Aleszczyk for their technical assistance.
Grant Lillian
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electron opaque stain in electron microscopy J Cell Biol 17: 208, 1963. Beutner EH, Sepulveda MR. Barnett EV: Quantitative studies of immunofluorescent staining. Bull WHO 39: 587, 1968. Churg J: Electron microscopic aspects of renal pathology, Structural Basis of Renal Disease (Becker EL, ed), New York, Harper & Row, 1966, p 132. Lerner RA, Glassock RJ, Dixon FJ: The role of antiglomerular basement membrane antibody in the pathogenesis of human glomerulonephritis. J Exp Med 126: 989,1967. Batting AJ, Brown AL, Divertie MB: The pulmonary lesion in a patient with Goodpasture’s syndrome as studied with the electron microscope. Amer J Clin Path 42: 387, 1964. Elliott A, Kuhn C: Idiopathic pulmonary hemosiderosis. Ultrastructural abnormalities in capillary walls. Amer Rev Resp Dis 102: 695, 1970. Weigle WO, Nakamura RM: Perpetuation of autoimmune thyroiditis and production of secondary renal lesions following periodic injections of aqueous preparations of altered thyroglobulin. Clin Exp lmmun 4: 645, 1969. Werner SC, Wegelius 0, Fierer JA, Hsu KC: Immunoglobulins (E.M,G) and complement in connective tissues of the thyroid in Graves’ disease. New Eng J Med 267: 421, 1972. Heimann P: Ultrastructure of human thyroid. Acta Endoer 53 (suppl 110): 1, 1966. Spear GS, Whitworth JM, Komgsmark BW: Hereditary nephritis with nerve deafness: immunofluorescent studies on the kidney, with a consideration of discordant immunoglobulin-complement immunofluorescent reactions. Amer J Med 49: 52, 1970. Gerber MA, Paronetto F: IgE in glomeruli of patients with nephrotic syndrome. The Lancet I: 1097, 1971. Sobel H, Geller J: Experimental thyroiditis in guinea pig: electron microscopy. Amer J Path 46: 149, 1965. Hall R: Immunologic aspects of thyroid function. New Eng J Med 266: 1204,1962.
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