- Email: [email protected]
Immunohistochemical study of intrathyroidal lymph follicles
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
lmmunohistochemical TAKESHI KASAJIMA,*
43, 117- 128 (1987)
Study of lntrathyroidal MITSUNORI
Lymph
YAMAKAWA,~ ANDYUTAKA
*Department of Pathology, Tohyo Women’s Medical College, 8-1 Kawadacho, Tokyo 162, Japan, and 7’Second Department of Pathology, School of Medicine, University. Zao-lida, Nishinomae, Yamagata 990-23, Japan
Follicles IMAI? Shinjuku-ku, Yamagata
Tissue samples from 76 cases of thyroid disease containing germinal centers (GCs) were selected from 396 cases of various thyroid diseases for immunohistochemical analysis of the GC and follicular dendritic cell (FDC). These 76 cases included 28 cases of thyroid cancer, 23 of chronic thyroiditis, 12 of nontoxic goiter, 8 of adenoma, and 5 of Graves’ disease. In many lymphoid GCs IgG, IgM and K and A light chains were detected with a lacy dendritic pattern. Only thyroglobulin (Tg), among the various thyroidassociated antigens sought, was stained in GCs and was found in the same distribution as immunoglobulins. Tg was found in all of the disease categories studied. Moreover, early components in the classical complement (C) pathway, that is, Clq. C4. C3c, C3d, and CS, were detected in almost all GCs in the thyroid tissues in a lacy dendritic pattern. C3d was intensely positive and was seen also by immunoelectron microscopy on the cell surface of FDCs. It is concluded that not only immunoglobulins but also Tg and early C components are closely related to the GC and FDC in the intrathyroidal lymph follicle. Q 1987 Academic Press, Inc.
INTRODUCTION
It is believed that secondary lymph follicles (LFs) generally appear in response to the invasion of heterologous antigens. They are not found in germ-free animals or in patients whose immunoglobulins are difficult to detect, as in agammaglobulinemia or related diseases. From the aspects of function and morphology, germinal centers (GCs) have been studied as a defense site against the invasion of various pathogens (1). However, secondary LFs are also found in many cases of autoimmune diseases, including Hashimoto’s thyroiditis and Graves’ disease (2). Immune complexes (ICs) may cause the appearance of secondary LFs with GC (3-7).
The existence of autoantibodies against thyroglobulin (Tg), microsomes and other antigens has been demonstrated in autoimmune thyroid diseases (8-13). Nevertheless, details of the histological distribution of Tg and complement (C) components in the GCs of thyroid tissues remain unknown (14). In this paper, we discuss the roles of immunoglobulins, Tg and C components, and follicular dendritic cells (FDCs) in the GCs in thyroid diseases. MATERIALS
AND METHODS
Tissues. Thyroid tissue samples (396) were obtained by surgical biopsy or thyroidectomy in the hospital of Yamagata University School of Medicine from 1977 to 1985 (Table 1). Dissected thyroid tissues were sliced approximately 5 mm thick and fixed in 10% Formalin. Eleven thyroid tissues with at least one secondary LF 117 0090-1229187 $1.50 Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.
118
KASAJIMA.
YALMAK>~UI\.
4ND
iMA
were trimmed and immediately snap fruzcn in l~ssue-lek 11 OC 1 compound (Miles Lab., Inc., Kankakee, IL) or immersed in periodate-lyxine-paraformaldehyde (PLP) fixative containing 2%. paraformaldehyde for 6 hr. rinsed in graded sucrose-phosphate-buffered saline. embedded into TissueTek II OCT cornpound, and stored at - 80°C until cryostat sectioning (15 I. Immunohistochemical reagents. Antisera were purchased r’rom the following companies. Rabbit anti-human IgG, IgM. IgE. K and A chains. Tg, thyrotropin (TSH), 3,5,5’-triiodothyronine (T,), thyroxine-binding globulin (TBG), and C3d antisera were obtained from DAK0 patts a/s, Copenhagen. Denmark. Rabbit anti-human IgA, IgD antisera, and goat anti-human c‘lq, C?c. C.C. and C3 activator antisera came from Hoechst Japan Co.. Tokyo. Japan. Rabbit anti-human thyroxine (T,) and calcitonin (CT) antisera were from Immulok, Inc., Carpinteria, California. Goat anti-human C3, C4, and C3b inactivator (C3b INA) antisera were from Kent Lab.. Inc., Malvern, Pennsylvania. Goat anti-human C9 and properdin were from Miles Scientific Lab., Naperville, Illinois. and anti-human C6. CX. and BIH globulin were from Cappel Lab.. Inc.. Malvern. Pennsylvania. As monoclonal antibodies, mouse anti-human dendritic reticulum cell-l (DRC-1) f 16. 17) and C3b receptor (C3bR) (18, 19) were purchased from DAK0 patts a/s. Mouse monoclonal anti-human Leu 1, Leu 2a. and Leu 3a were from Becton-Dickinson, Monoclonal Center, Inc., Mountain View, California. and OKT 3, OKT 4. OKT 6, and OKT 8 were from Ortho Diagnostics System Co., Tokyo. Japan. As secondary antisera, horseradish peroxidase (HRP)-conjugated swine anti-rabbit lg (DAK0 patts a/s), HRP-conjugated F(ab’), fragment rabbit anti-goat 1gG (Cappel), HRP-conjugated F(ab’), fragment sheep anti-mouse Ig (Amersham Japan Co., Tokyo, Japan), and HRP-conjugated goat anti-mouse IgM (TAGO, Inc., Burlingame, CA) antisera were used. 3.?‘-Diaminobenzidine tetrahydrochloride (Dotite) was obtained from Dojin Chemicals, Tokyo. Japan. Preparation of HRP-conjuguted F(ab’), fragmerlt. Rabbit anti-human IgG. IgM, K chain, Tg, C3d antisera, and goat anti-human Clq. C5 antisera were hydrolyzed to F(ab’), fragment and conjugated with HRP. as described previously by Farr and Nakane (20). Tissue preparation. Formalin-fixed and paraffin-embedded tissue sections were prepared 4 km thick and stained with hematoxylin-eosin and stained immunohistochemically. Frozen tissues were cut into 4 km by cryostat (Leitz, Germany) and used for immunohistochemical staining. Immunoperoxiduse procedure. All antisera, as mentioned above. were applied to cryostat sections. The antisera against immunoglobulins (IgG, IgM. IgA, IgD, IgE, K and h chains) were applied to paraffin sections. All antisera were tested over a range of dilutions on tissue sections and subsequently used at their optimal concentrations (immunoglobulins, l/400-800; thyroid-associated antigens. 11400: C components, 11200-400; monoclonal antisera. l/200-400; secondary antisera, l/50- 100). Direct and indirect immunoperoxidase staining was used for light microscopy, and HRP-conjugated F(ab’), fragment IgG antisera were used for immunoelectron microscopy, as described previously (20). Control studies were simultaneously carried out as follows. (I) lnstead of primary antiserum, normal rabbit, goat. or mouse serum was used. (2) Nonspecific
INTRATHYROIDAL
LYMPH
119
FOLLICLE
staining was checked by omitting primary antiserum. (3) Primary antiserum was absorbed by corresponding purified antigen. Simultaneously, the specific reaction directed to each pure antigen was checked by using the double immunodiffusion (Ouchterlony) method. (4) Secondary antiserum was omitted. (5) Endogenous peroxidase activity was checked by using only the diaminobenzidine reaction. RESULTS
Frequency of GCs’ appearance in various thyroid diseases. As shown in Table 1, GCs appeared more frequently in chronic thyroiditis or in Graves’ disease, that is, in so-called autoimmune thyroid disease, than in the others. Light microscopic features of GCs in thyroid tissues. In chronic thyroiditis, many hyperplastic secondary LFs were found in the thyroid stromal tissues (Fig. 1). Secondary LFs often surrounded tumor nodules of thyroid cancer or adenoma. A large number of medium- and large-sized lymphocytes (germinocytes and germinoblasts), many follicular dendritic cells (FDCs) with euchromatic and horseshoe-shaped nuclei (21), and one or more macrophages with evidence of phagocytic activity (tingible body/starry-sky macrophage) were seen in the GCs. Localization of immunoglobulins in the GCs. In 76 cases with secondary LFs among 396 cases, localization of immunoglobulins was studied using the indirect immunoperoxidase procedure with Formalin-fixed paraffin sections. Histologic findings in these 76 cases are shown in Table 1. IgG, IgM, and IgA were stained in many GCs with some differences in the intensity of positivity and number of positive cells. On the other hand, IgD and IgE were not stained in most GCs. IgG and IgM were found more often in the tissues from chronic thyroiditis and Graves’ disease than in those of other categories (Fig. 2a). Within the GCs, IgA was rarely stained in a lacy dendritic pattern, and most of those positive for IgG showed a scattered pattern. By immunoelectron microscopy, IgM localized mainly in the intercellular space and in the cytoplasm and was especially abundant on the cell surface of the labyrinth structure of FDCs (Fig. 2b). IgM also was detected in the perinuclear cistern and rough endoplasmic reticulum of lymphoid cells of GCs. IgG localized in a manner similar to IgM. Generally, the distribution of both K and A chains in the GCs was similar to that TABLE
1
TOTALCASESANDCASESWITHSECONDAR~LYMPH
Histologic types Cancer Chronic thyroiditis Nontoxic goiter Adenoma Graves’ disease Others Total
FOLLICLE
With secondary lymph follicle
Total cases
Cases
(%I
Male
Female
M/F
97 28 113 128 12 18 396
28 23 12 8 5 0 76
28.9 82.1 10.6 6.3 41.7 0 19.2
4 2 0 2 0 0 8
24 21 12 6 5 0 68
l/6 l/IO 113 118.5
FIG.
1. Histologic
with hyperplastic cell infiltration. Hematoxylin
appearance
of tissue
from
germinal centers. ~48. (b) mainly composed of lymphocytes. and eosin.
chronic
thyroiditi\
High-power pla\m;l
vlen cell\.
(a) 7‘hrtt~
i\mphotd
of I,~I \howmg and destro\eti
nl;i\\l\c
follicle\
‘I,!”
intl;lmmato> . _‘X’ follicle\
INTRATHYROIDAL
LYMPH
FOLLICLE
121
of IgM, and there were no noticeable differences in the degree of intensity or in the quantity of positive cells between the light chains. The staining for immunoglobulins was checked for specificity by the abovementioned control studies. No immunostaining was found when the first and/or second antisera were omitted. Localization of thyroid-associated antigens in the GCs. Light and electron microscopic immunostaining of Tg was recognized in the intercellular space and on the cell surface in the GCs. In particular, electron-positive products for anti-Tg antiserum were recognized attaching to the surface of the labyrinth structure of FDCs, but there were never reaction products in the cytoplasm of any germinocytes, germinoblasts, and FDCs (Fig. 3). The immunostaining of Tg was checked for specificity by the above-mentioned control studies. No immunostaining was found when primary and/or secondary antisera were omitted or when anti-Tg antiserum, after being absorbed with human Tg, was used as the primary antiserum. Moreover. no cross-reaction was found between rabbit anti-human IgG, IgM, and IgA antisera and human Tg in a study using the immunodiffusion technique. On the other hand, the other five thyroid-associated antigens were not stained in any GCs. These results are summarized in Table 2. Tg was frequently recognized by light microscopic observation in tumor cells which were distributed in the thyroid tumor tissues (thyroid adenoma and cancer).
FIG. 2. Immunostaining of IgM in chronic thyroiditis. (a) Distinct lacy dendritic pattern within the germinal center. Formalin-fixed and paraffin-embedded section, indirect immunoperoxidase staining, counterstained with methylgreen. x 120. (b) Immunoelectron photomicrograph showing reaction product on the surface of the labyrinth structure of a follicular dendritic cell (arrows). x 4700.
Loccrli:ution of’C c’omponc’tlts~lrrd C3hR in l11cj (;Cs. The I 1 casts (mentioned above) were examined for reaction to various antisera against C components and C3bR. Clq. C3c, C3d (Fig. 4a), C3b INA, C5, and C3bR were clearly present in a lacy dendritic pattern within almost ail GCs. Clq, C3d. and C3bR reacted more strongly than the others. On the other hand, late components examined in the C-activating pathway. that is, C6. 0. and C9. did not react except for a feu mononuclear cells, which were considered to be macrophages in the GCq. There was no positive staining for alternative pathway C components (C3 activator and properdin). C4 was often stained, but C3 and PlH globulin were not recognized. As seen with immunoelectron microscopy. Ctq. C3d (Fig. 4b). C.S, and C3bR (Fig. 5) reacted on the cell surface and/or in the intercellular space. Moreover. C3d was stained more strongly on the surface of FDCs than on that of germinocytes or germinoblasts. C components, however, were never stained positively in the cell organelles of germinocytes, germinoblasts. or FDCs. Furthermore, Clq, C3d, and C5 were recognized in a lacy dendritic pattern similar to that of IgG, IgM, or Tg in the same GCs. No immunostaining of C components (Clq. C4. C3c: C3d, C3b INA, and C5) was found when primary and/or secondary antisera were omitted. Furthermore. no cross-reactivity was found by immunodiffusion between anti-human C component antisera and Tg.
INTRATHYROIDAL
LYMPH
123
FOLLICLE
Localization of FDC and T-cell subsets in the GCs. Mouse monoclonal antibodies against human DRC-1 and T-cell subsets were applied to cryostat sections of the above-mentioned 1 I cases. DRC-1 showed a strong lacy dendritic pattern within all GCs (Fig. 6). The reaction products for anti-DRC-1 antiserum were clearly found by immunoelectron microscopy, especially attached to the cell membranes of FDCs and their cell processes. Monoclonal antibodies against human T-cell subsets reacted on fewer cell surfaces in GCs. Sometimes a striking crescentic accumulation of positive cells was also noted to occur at the junction of the inner aspect of the lymphocyte mantle and the outer aspect of the GCs. The number of Leu 3a and OKT 4-positive cells was slightly less than that obtained with Leu 1 and OKT 3 but more than that obtained with Leu 2a and OKT 8. Within the GCs of the thyroid tissues, the ratio of Leu 2a/Leu 3a or OKT 8/OKT4 varied from 1:lS to 1:4.0, and the helper/inducer T cells were dominant in comparison to suppressoricytotoxic T cells. OKT 6-positive cells, however, were not found. The results of our immunohistochemical study on the GCs in 76 cases with secondary LFs are summarized in Table 3. DISCUSSION
Observations in the present and earlier studies show that the appearance of GCs in thyroid tissue is not peculiar to autoimmune disease, because GCs were also found in the tissues of thyroid cancer, adenoma, and goiter. GCs were frequently found adjacent to tumor nodules and also within tumor nodules. Nevertheless, GCs appeared more frequently in autoimmune thyroid diseases. It has been reported that thyroid diseases are predominant in females (2, 1 1), and cases with secondary LFs were remarkably dominant in females (68 out of 76 cases) in our study. Table 3 summarizes the results of our immunohistochemical study on GCs in thyroid diseases. Within many GCs of thyroid tissues, IgG, IgM, and both light TABLE 2 LOCALIZATIONOFTHYROIDTISSUEANTIGENSINTHEGERMINALCENTERSOFTHYROID Cases Graves’
disease
Cancer
Chronic Adenoma
thyroiditis
1 2 3 4 5 1 2 3 1 2 I
TISSUES
Tg
TSH
T3
T4
TBG
+ + + 7
-
-
-
-
+ + + + +
Note. Tg, thyroglobulin; TSH, thyrotropin; binding globulin; CT, calcitonin; + , distinct mine (blurred stain).
T,, triiodothyronine; positive; - , negative:
? -
CT
? -
T,, thyroxine; TBG, thyroxine?, positivity was difficult to deter-
124
KASAJIMA.
\rAMAhAW’i.
%.\I.,
!&I.\:
chains were distributed in a lacy dendritlc pattern. which \$,I- iiclccit‘d i>! li.iimunoelectron microscopy on the cell surface and In tht’ intcrccllular hpacc, Also. Igll and IgE. hoL\IgA was stained mainly in a scattered pattern in man)’ c;ws ever, were almost never seen in GC\ Autoantibodies against Tg, microsome\. i , ot- I‘,. and lhh rood follicuiar clcments have been found in the sera of patients with thyroid disease> ! I l- 1.1). Anti-Tg and microsome antibodies have been detected more frequently in Hashimoto’s thyroiditis and Graves’ disease. Moreover. the deposition of’ ICs has been identified in the basement membrane beneath the colloid epithelia and vasculature in thyroid tissues (22-26). Therefore. it seemsthat autoantibodie\ andior 1Cs closely relate to the pathogenesis of these thyroid diseases. in thi$ study. Tg. but not TSH. T,, T,, TBG, and CT, was stained in many case\ in a lacy dendritic pattern within the GCs in thyroid tissues. Considering that in the same case%IgG, IgM, and Tg were stained in a similar dendritic fashion, it may be presumed that the Tg-anti-Tg antibody IC exists in the GCs. Tdkeda and Krisq 122) and Mariotti et al. (24) have reported on the presence of IC in patients‘ sera. Furthermore. Albini t’z rrl. (27) have recently reported that the immunoperoxidase reaction was intensified by dissociation of the endogenous immune complex. followed by reaction of the tissue with added anti-Tg. and have suggested the existence of an
FIG. 4. Same case as shown in Fig. 2. Immunostaining of complement component C3d. (a) Distinct lacy dendritic staining within the germinal center. Periodate-lysine-paraformaldehyde-fixed cryostat section, counterstained with methylgreen. x 125. (b) Immunoelectron photomicrograph showing abundant reaction product on the cell surface of the labyrinth structure of follicular dendritic cell. x 3700.
INTRATHYROIDAL
LYMPH
FOLLICLE
125
FIG. 5. Immunoperoxidase staining for C3b receptor in the tissue surrounding an adenoma nodule. Electron photograph showing positive staining on the cell surface of a follicular dendritic cell. x 7500.
endogenous Tg-anti-Tg IC. If it is assumed that ICs play an important role in the appearance of GCs, Tg-anti-Tg IC should be more important than the other antibodies (TSH, T,, and T4). Many C receptors on the cell surface have been reported, i.e., Clq, C3a, C3b, iC3b, C3d, C5, and PIH globulin receptors (28). Among these receptors, C3bR and C3d receptor (C3dR) have been considered to play the most important role in the binding of IC. The presence of C3bR on the cell surface of human erythrocytes, monocyte/macrophages, B cells, and certain T cells has been accepted (18, 29, 30). Furthermore, it is also accepted that C3dR is present on the cell surface of human monocyte/macrophages and B cells (30,3 1). Recently, Gerdes and Stein (32) reported that there are many C3 receptors on the DRC in the GCs, and more C3 receptors were found on the DRCs than on the circumscribed B cells. In the present study many GCs were stained in a lacy dendritic pattern with the antisera against Clq, C3c, C3d, C3b INA, and C3bR, and some were also detected on the cell surface of germinocytes, germinoblasts, or FDCs and in the intercellular space within the GCs. Using immunohistochemical methods we recently identified the presence of C3dR on the cell surface in GCs (33). ICs bind to the cell
126
FI(s. 6. Immunosti photl ograph showing tion. indirect immune
KASAJIMA.
YAMAkAU.I\.
4%11) lM.4)
Light micros :opic fixed cryostat sec-
surface by the binding between C3d and C3dK preventing acll\,ation of C5. and then IC binding to C3 is retained on the cell surface of FIX for a long period under more stable conditions. As Klaus rr rrl. (341 mentioned concerning the retention of ICs and presenting of antigens to germinocytex. FIX may play an important role in the immune response within the CJC‘S. On the other hand, late components (C6. C8. and 0) and alternative pathway components (C3 activator and properdin) were not detected within the GCs. It is suggested, however, that late C components may participate under conditions involving the solubilization of ICs (35-37). In the present study we investigated the distribution of T-cell subsets within the GCs of thyroid tissues. Poppema et rrl. (38) reported T-cell subsets within the GCs of human lymph nodes, but there have been no reports of T-cell subsets within the GCs in thyroid tissues. Our results resemble those of Poppema. The number of Leu 3a and OKT 4-positive helper/inducer cells was more than that of Leu 2a and OKT 8 suppressor/cytotoxic cells, and sometimes a group of these positive cells was seen at the periphery of GCs. It is reasonable to assume that T helper inducer cells play an important role in the formation of GCs and in the
INTRATHYROIDAL
127
LYMPH FOLLICLE
TABLE 3 REACTIVITYTOVARIOUSANTISERAWITHINTHEGERMINALCENTERSIN~~CASESWITH SECONDARYLYMPH FOLLICLES Antisera Immunoglobulins 1s kM Id K free chain A free chain Thyroid associated Thyroglobulin Thyroxine Triiodothyronine Thyrotropin Thyroxine-binding globulin Monoclonal antisera Dendritic reticulum cell 1 C3b receptor Leu l,OKT3 Leu 2a. OKT 8 Leu 3a, OKT 4 OKT6
Reactivity
++ +++ + +++ +++ +++ -
+++ +++ + + +
Antisera Complement components Ch c4 c3 c3c C3d C5 C6 C8 c9 C3 activator Properdin Regulatory Protein C3b inactivator BlH globulin
Reactivity ++ -I+ + +++ + -
+ -
Note. + + + , Strongly positive; + + , moderately positive; + . weakly positive; - , negative. These 76 cases comprised 28 with cancer, 23 with chronic thyroiditis, 12 with nontoxic goiter, 8 with adenoma. and 5 with Graves’ disease.
induction of B-cell differentiation within the intrathyroidal secondary LFs as within the other nodal secondary LFs. Moreover, it seems that T cells, B cells, tingible body macrophages, and FDCs are all required for immune reaction within the GCs. ACKNOWLEDGMENTS This work was supported by Grant-in-Aid for General Research (B) No. 59480142, from the Ministry of Education, Science and Culture, Japan. The authors thank Professor Akira Kajita and Professor Mizu Kojima, Tokyo Women’s Medical College, for helpful advice and discussion and also Associate Professor J. Petrich Barron, St. Marianna University School of Medicine. for his revision of the manuscript.
REFERENCES 1. Imai, Y., Terashima, K., Matsuda, M., Maeda, K., and Kasajima, T., Recent Adv. RES Res. 21, 51, 1983. 2. Meissner, W. A., In “The Thyroid: A Fundamental and Clinical Test” (S. C. Werner, and S. H. Ingbar, Eds.), pp. 444-479, Harper & Row, New York, 1978. 3. Clagett, J. A., Wilson, C. B., and Weigle, W. A., J. Exp. Med. 140, 1439, 1974. 4. Kalderon, A. E., and Bogaars, H. A., Amer. J. Med. 63, 729, 1977. 5. Fujiwara, H., Torisu. M., Koitabashi, Y., Baba. T., and Esaki. H., C&n. Immunol. Immunopathol. 19, 98, 1981.
IX
KASAJIMA.
1 ,\MAK,411
t3.
.\hl.i
IMhi
6. Fujiwara. H.. lb1-1>11. 34.. Sugisaki. ‘I’.. anti Ohant). ii ~ i !i; fiJi,,li,,ia ,I !,:,/,~,!/i,~;?,!!i!i’: IY. i<“i 19x1. 7. Yagi, Y.. .-lc’t 41. \hital\c. 5. ;~nci I\h~ha\\;! f ! (‘!!I! iYlIdi~( r’lll;il Metub. 52. 739. t9x I. I I. VolpC. R.. //I “Auto-immunity in the Endocrine S)\lem: .4uto~rnmunrt~ /II Th!~rord L>txa\e. pp. 19- I 1 I ( Springer-Verlag. Berlin/New York, 19x1 I?. Davies. T. F., and Bernardo. E. D.. 1,~ “Autoimmune F.ndocrme Dlac,l\e I hbrold Autoantl bodies and Disease. An Overview” (T. F. Davies. Ed.). pp. 117- 137. Wiley-Interccience. Nen York. 1983. 13. Weetmnn. ‘4. P.. and McGregor. A. M.. Elrc~ho,?~. (‘~roc.hc~r~~. 22, 1077. 1974. 16. Parwaresch, M. R.. Radzun H. J.. Feller. :\. C.. Peter\. K. P... and Hausmann. M. L.. .I. Inrtmrnol. 131. 1719. 1983. 17. Naime. M.. Gerdes. J.. Abdulaziz, Z. Stein. H.. and Mason. I). Y.. .I. ( /i/r. Prrthoi, 36. 167. 1983. 18. Fearon. 1). T.. .I. EL-II. Med. 152, 20. 1980. 19. Gerdes. J.. Nami. M.. Mason, D. Y.. and Stein. H.. fn~~rnrnol~~,~ 45, 645. 1981. 20. Farr, A. G.. and Nakane. P. K.. J. If,~l,zrrrto/. Mrllwd.c 47, 179. 1981. 21. Tew. J. G.. Thorbecke. G. J.. and Steinman. R. M.. .I. RES 31. 371. 1982. 22. Takeda. Y.. and Kriss. J. P.. J. Clirl. Endocriuc,l. Mertrh. 44. 46. 1977. 23. Pinchera. A.. Mariotti. S.. Vitti. P.. Tosi, M.. Grasao. L.. Pacini. F.. Buti. R.. and Baschieri. L.. J. C/in. Endocrinol. Metcth. 45. 1077. 1977. 24. Mariotti. S.. DeCroot. L. J.. Scarborough. D.. and Medof. M. E.. .I. C‘/ifl. tnd
July 8. 1986: accepted
with
revision
January
6. 19X7
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
lmmunohistochemical TAKESHI KASAJIMA,*
43, 117- 128 (1987)
Study of lntrathyroidal MITSUNORI
Lymph
YAMAKAWA,~ ANDYUTAKA
*Department of Pathology, Tohyo Women’s Medical College, 8-1 Kawadacho, Tokyo 162, Japan, and 7’Second Department of Pathology, School of Medicine, University. Zao-lida, Nishinomae, Yamagata 990-23, Japan
Follicles IMAI? Shinjuku-ku, Yamagata
Tissue samples from 76 cases of thyroid disease containing germinal centers (GCs) were selected from 396 cases of various thyroid diseases for immunohistochemical analysis of the GC and follicular dendritic cell (FDC). These 76 cases included 28 cases of thyroid cancer, 23 of chronic thyroiditis, 12 of nontoxic goiter, 8 of adenoma, and 5 of Graves’ disease. In many lymphoid GCs IgG, IgM and K and A light chains were detected with a lacy dendritic pattern. Only thyroglobulin (Tg), among the various thyroidassociated antigens sought, was stained in GCs and was found in the same distribution as immunoglobulins. Tg was found in all of the disease categories studied. Moreover, early components in the classical complement (C) pathway, that is, Clq. C4. C3c, C3d, and CS, were detected in almost all GCs in the thyroid tissues in a lacy dendritic pattern. C3d was intensely positive and was seen also by immunoelectron microscopy on the cell surface of FDCs. It is concluded that not only immunoglobulins but also Tg and early C components are closely related to the GC and FDC in the intrathyroidal lymph follicle. Q 1987 Academic Press, Inc.
INTRODUCTION
It is believed that secondary lymph follicles (LFs) generally appear in response to the invasion of heterologous antigens. They are not found in germ-free animals or in patients whose immunoglobulins are difficult to detect, as in agammaglobulinemia or related diseases. From the aspects of function and morphology, germinal centers (GCs) have been studied as a defense site against the invasion of various pathogens (1). However, secondary LFs are also found in many cases of autoimmune diseases, including Hashimoto’s thyroiditis and Graves’ disease (2). Immune complexes (ICs) may cause the appearance of secondary LFs with GC (3-7).
The existence of autoantibodies against thyroglobulin (Tg), microsomes and other antigens has been demonstrated in autoimmune thyroid diseases (8-13). Nevertheless, details of the histological distribution of Tg and complement (C) components in the GCs of thyroid tissues remain unknown (14). In this paper, we discuss the roles of immunoglobulins, Tg and C components, and follicular dendritic cells (FDCs) in the GCs in thyroid diseases. MATERIALS
AND METHODS
Tissues. Thyroid tissue samples (396) were obtained by surgical biopsy or thyroidectomy in the hospital of Yamagata University School of Medicine from 1977 to 1985 (Table 1). Dissected thyroid tissues were sliced approximately 5 mm thick and fixed in 10% Formalin. Eleven thyroid tissues with at least one secondary LF 117 0090-1229187 $1.50 Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.
118
KASAJIMA.
YALMAK>~UI\.
4ND
iMA
were trimmed and immediately snap fruzcn in l~ssue-lek 11 OC 1 compound (Miles Lab., Inc., Kankakee, IL) or immersed in periodate-lyxine-paraformaldehyde (PLP) fixative containing 2%. paraformaldehyde for 6 hr. rinsed in graded sucrose-phosphate-buffered saline. embedded into TissueTek II OCT cornpound, and stored at - 80°C until cryostat sectioning (15 I. Immunohistochemical reagents. Antisera were purchased r’rom the following companies. Rabbit anti-human IgG, IgM. IgE. K and A chains. Tg, thyrotropin (TSH), 3,5,5’-triiodothyronine (T,), thyroxine-binding globulin (TBG), and C3d antisera were obtained from DAK0 patts a/s, Copenhagen. Denmark. Rabbit anti-human IgA, IgD antisera, and goat anti-human c‘lq, C?c. C.C. and C3 activator antisera came from Hoechst Japan Co.. Tokyo. Japan. Rabbit anti-human thyroxine (T,) and calcitonin (CT) antisera were from Immulok, Inc., Carpinteria, California. Goat anti-human C3, C4, and C3b inactivator (C3b INA) antisera were from Kent Lab.. Inc., Malvern, Pennsylvania. Goat anti-human C9 and properdin were from Miles Scientific Lab., Naperville, Illinois. and anti-human C6. CX. and BIH globulin were from Cappel Lab.. Inc.. Malvern. Pennsylvania. As monoclonal antibodies, mouse anti-human dendritic reticulum cell-l (DRC-1) f 16. 17) and C3b receptor (C3bR) (18, 19) were purchased from DAK0 patts a/s. Mouse monoclonal anti-human Leu 1, Leu 2a. and Leu 3a were from Becton-Dickinson, Monoclonal Center, Inc., Mountain View, California. and OKT 3, OKT 4. OKT 6, and OKT 8 were from Ortho Diagnostics System Co., Tokyo. Japan. As secondary antisera, horseradish peroxidase (HRP)-conjugated swine anti-rabbit lg (DAK0 patts a/s), HRP-conjugated F(ab’), fragment rabbit anti-goat 1gG (Cappel), HRP-conjugated F(ab’), fragment sheep anti-mouse Ig (Amersham Japan Co., Tokyo, Japan), and HRP-conjugated goat anti-mouse IgM (TAGO, Inc., Burlingame, CA) antisera were used. 3.?‘-Diaminobenzidine tetrahydrochloride (Dotite) was obtained from Dojin Chemicals, Tokyo. Japan. Preparation of HRP-conjuguted F(ab’), fragmerlt. Rabbit anti-human IgG. IgM, K chain, Tg, C3d antisera, and goat anti-human Clq. C5 antisera were hydrolyzed to F(ab’), fragment and conjugated with HRP. as described previously by Farr and Nakane (20). Tissue preparation. Formalin-fixed and paraffin-embedded tissue sections were prepared 4 km thick and stained with hematoxylin-eosin and stained immunohistochemically. Frozen tissues were cut into 4 km by cryostat (Leitz, Germany) and used for immunohistochemical staining. Immunoperoxiduse procedure. All antisera, as mentioned above. were applied to cryostat sections. The antisera against immunoglobulins (IgG, IgM. IgA, IgD, IgE, K and h chains) were applied to paraffin sections. All antisera were tested over a range of dilutions on tissue sections and subsequently used at their optimal concentrations (immunoglobulins, l/400-800; thyroid-associated antigens. 11400: C components, 11200-400; monoclonal antisera. l/200-400; secondary antisera, l/50- 100). Direct and indirect immunoperoxidase staining was used for light microscopy, and HRP-conjugated F(ab’), fragment IgG antisera were used for immunoelectron microscopy, as described previously (20). Control studies were simultaneously carried out as follows. (I) lnstead of primary antiserum, normal rabbit, goat. or mouse serum was used. (2) Nonspecific
INTRATHYROIDAL
LYMPH
119
FOLLICLE
staining was checked by omitting primary antiserum. (3) Primary antiserum was absorbed by corresponding purified antigen. Simultaneously, the specific reaction directed to each pure antigen was checked by using the double immunodiffusion (Ouchterlony) method. (4) Secondary antiserum was omitted. (5) Endogenous peroxidase activity was checked by using only the diaminobenzidine reaction. RESULTS
Frequency of GCs’ appearance in various thyroid diseases. As shown in Table 1, GCs appeared more frequently in chronic thyroiditis or in Graves’ disease, that is, in so-called autoimmune thyroid disease, than in the others. Light microscopic features of GCs in thyroid tissues. In chronic thyroiditis, many hyperplastic secondary LFs were found in the thyroid stromal tissues (Fig. 1). Secondary LFs often surrounded tumor nodules of thyroid cancer or adenoma. A large number of medium- and large-sized lymphocytes (germinocytes and germinoblasts), many follicular dendritic cells (FDCs) with euchromatic and horseshoe-shaped nuclei (21), and one or more macrophages with evidence of phagocytic activity (tingible body/starry-sky macrophage) were seen in the GCs. Localization of immunoglobulins in the GCs. In 76 cases with secondary LFs among 396 cases, localization of immunoglobulins was studied using the indirect immunoperoxidase procedure with Formalin-fixed paraffin sections. Histologic findings in these 76 cases are shown in Table 1. IgG, IgM, and IgA were stained in many GCs with some differences in the intensity of positivity and number of positive cells. On the other hand, IgD and IgE were not stained in most GCs. IgG and IgM were found more often in the tissues from chronic thyroiditis and Graves’ disease than in those of other categories (Fig. 2a). Within the GCs, IgA was rarely stained in a lacy dendritic pattern, and most of those positive for IgG showed a scattered pattern. By immunoelectron microscopy, IgM localized mainly in the intercellular space and in the cytoplasm and was especially abundant on the cell surface of the labyrinth structure of FDCs (Fig. 2b). IgM also was detected in the perinuclear cistern and rough endoplasmic reticulum of lymphoid cells of GCs. IgG localized in a manner similar to IgM. Generally, the distribution of both K and A chains in the GCs was similar to that TABLE
1
TOTALCASESANDCASESWITHSECONDAR~LYMPH
Histologic types Cancer Chronic thyroiditis Nontoxic goiter Adenoma Graves’ disease Others Total
FOLLICLE
With secondary lymph follicle
Total cases
Cases
(%I
Male
Female
M/F
97 28 113 128 12 18 396
28 23 12 8 5 0 76
28.9 82.1 10.6 6.3 41.7 0 19.2
4 2 0 2 0 0 8
24 21 12 6 5 0 68
l/6 l/IO 113 118.5
FIG.
1. Histologic
with hyperplastic cell infiltration. Hematoxylin
appearance
of tissue
from
germinal centers. ~48. (b) mainly composed of lymphocytes. and eosin.
chronic
thyroiditi\
High-power pla\m;l
vlen cell\.
(a) 7‘hrtt~
i\mphotd
of I,~I \howmg and destro\eti
nl;i\\l\c
follicle\
‘I,!”
intl;lmmato> . _‘X’ follicle\
INTRATHYROIDAL
LYMPH
FOLLICLE
121
of IgM, and there were no noticeable differences in the degree of intensity or in the quantity of positive cells between the light chains. The staining for immunoglobulins was checked for specificity by the abovementioned control studies. No immunostaining was found when the first and/or second antisera were omitted. Localization of thyroid-associated antigens in the GCs. Light and electron microscopic immunostaining of Tg was recognized in the intercellular space and on the cell surface in the GCs. In particular, electron-positive products for anti-Tg antiserum were recognized attaching to the surface of the labyrinth structure of FDCs, but there were never reaction products in the cytoplasm of any germinocytes, germinoblasts, and FDCs (Fig. 3). The immunostaining of Tg was checked for specificity by the above-mentioned control studies. No immunostaining was found when primary and/or secondary antisera were omitted or when anti-Tg antiserum, after being absorbed with human Tg, was used as the primary antiserum. Moreover. no cross-reaction was found between rabbit anti-human IgG, IgM, and IgA antisera and human Tg in a study using the immunodiffusion technique. On the other hand, the other five thyroid-associated antigens were not stained in any GCs. These results are summarized in Table 2. Tg was frequently recognized by light microscopic observation in tumor cells which were distributed in the thyroid tumor tissues (thyroid adenoma and cancer).
FIG. 2. Immunostaining of IgM in chronic thyroiditis. (a) Distinct lacy dendritic pattern within the germinal center. Formalin-fixed and paraffin-embedded section, indirect immunoperoxidase staining, counterstained with methylgreen. x 120. (b) Immunoelectron photomicrograph showing reaction product on the surface of the labyrinth structure of a follicular dendritic cell (arrows). x 4700.
Loccrli:ution of’C c’omponc’tlts~lrrd C3hR in l11cj (;Cs. The I 1 casts (mentioned above) were examined for reaction to various antisera against C components and C3bR. Clq. C3c, C3d (Fig. 4a), C3b INA, C5, and C3bR were clearly present in a lacy dendritic pattern within almost ail GCs. Clq, C3d. and C3bR reacted more strongly than the others. On the other hand, late components examined in the C-activating pathway. that is, C6. 0. and C9. did not react except for a feu mononuclear cells, which were considered to be macrophages in the GCq. There was no positive staining for alternative pathway C components (C3 activator and properdin). C4 was often stained, but C3 and PlH globulin were not recognized. As seen with immunoelectron microscopy. Ctq. C3d (Fig. 4b). C.S, and C3bR (Fig. 5) reacted on the cell surface and/or in the intercellular space. Moreover. C3d was stained more strongly on the surface of FDCs than on that of germinocytes or germinoblasts. C components, however, were never stained positively in the cell organelles of germinocytes, germinoblasts. or FDCs. Furthermore, Clq, C3d, and C5 were recognized in a lacy dendritic pattern similar to that of IgG, IgM, or Tg in the same GCs. No immunostaining of C components (Clq. C4. C3c: C3d, C3b INA, and C5) was found when primary and/or secondary antisera were omitted. Furthermore. no cross-reactivity was found by immunodiffusion between anti-human C component antisera and Tg.
INTRATHYROIDAL
LYMPH
123
FOLLICLE
Localization of FDC and T-cell subsets in the GCs. Mouse monoclonal antibodies against human DRC-1 and T-cell subsets were applied to cryostat sections of the above-mentioned 1 I cases. DRC-1 showed a strong lacy dendritic pattern within all GCs (Fig. 6). The reaction products for anti-DRC-1 antiserum were clearly found by immunoelectron microscopy, especially attached to the cell membranes of FDCs and their cell processes. Monoclonal antibodies against human T-cell subsets reacted on fewer cell surfaces in GCs. Sometimes a striking crescentic accumulation of positive cells was also noted to occur at the junction of the inner aspect of the lymphocyte mantle and the outer aspect of the GCs. The number of Leu 3a and OKT 4-positive cells was slightly less than that obtained with Leu 1 and OKT 3 but more than that obtained with Leu 2a and OKT 8. Within the GCs of the thyroid tissues, the ratio of Leu 2a/Leu 3a or OKT 8/OKT4 varied from 1:lS to 1:4.0, and the helper/inducer T cells were dominant in comparison to suppressoricytotoxic T cells. OKT 6-positive cells, however, were not found. The results of our immunohistochemical study on the GCs in 76 cases with secondary LFs are summarized in Table 3. DISCUSSION
Observations in the present and earlier studies show that the appearance of GCs in thyroid tissue is not peculiar to autoimmune disease, because GCs were also found in the tissues of thyroid cancer, adenoma, and goiter. GCs were frequently found adjacent to tumor nodules and also within tumor nodules. Nevertheless, GCs appeared more frequently in autoimmune thyroid diseases. It has been reported that thyroid diseases are predominant in females (2, 1 1), and cases with secondary LFs were remarkably dominant in females (68 out of 76 cases) in our study. Table 3 summarizes the results of our immunohistochemical study on GCs in thyroid diseases. Within many GCs of thyroid tissues, IgG, IgM, and both light TABLE 2 LOCALIZATIONOFTHYROIDTISSUEANTIGENSINTHEGERMINALCENTERSOFTHYROID Cases Graves’
disease
Cancer
Chronic Adenoma
thyroiditis
1 2 3 4 5 1 2 3 1 2 I
TISSUES
Tg
TSH
T3
T4
TBG
+ + + 7
-
-
-
-
+ + + + +
Note. Tg, thyroglobulin; TSH, thyrotropin; binding globulin; CT, calcitonin; + , distinct mine (blurred stain).
T,, triiodothyronine; positive; - , negative:
? -
CT
? -
T,, thyroxine; TBG, thyroxine?, positivity was difficult to deter-
124
KASAJIMA.
\rAMAhAW’i.
%.\I.,
!&I.\:
chains were distributed in a lacy dendritlc pattern. which \$,I- iiclccit‘d i>! li.iimunoelectron microscopy on the cell surface and In tht’ intcrccllular hpacc, Also. Igll and IgE. hoL\IgA was stained mainly in a scattered pattern in man)’ c;ws ever, were almost never seen in GC\ Autoantibodies against Tg, microsome\. i , ot- I‘,. and lhh rood follicuiar clcments have been found in the sera of patients with thyroid disease> ! I l- 1.1). Anti-Tg and microsome antibodies have been detected more frequently in Hashimoto’s thyroiditis and Graves’ disease. Moreover. the deposition of’ ICs has been identified in the basement membrane beneath the colloid epithelia and vasculature in thyroid tissues (22-26). Therefore. it seemsthat autoantibodie\ andior 1Cs closely relate to the pathogenesis of these thyroid diseases. in thi$ study. Tg. but not TSH. T,, T,, TBG, and CT, was stained in many case\ in a lacy dendritic pattern within the GCs in thyroid tissues. Considering that in the same case%IgG, IgM, and Tg were stained in a similar dendritic fashion, it may be presumed that the Tg-anti-Tg antibody IC exists in the GCs. Tdkeda and Krisq 122) and Mariotti et al. (24) have reported on the presence of IC in patients‘ sera. Furthermore. Albini t’z rrl. (27) have recently reported that the immunoperoxidase reaction was intensified by dissociation of the endogenous immune complex. followed by reaction of the tissue with added anti-Tg. and have suggested the existence of an
FIG. 4. Same case as shown in Fig. 2. Immunostaining of complement component C3d. (a) Distinct lacy dendritic staining within the germinal center. Periodate-lysine-paraformaldehyde-fixed cryostat section, counterstained with methylgreen. x 125. (b) Immunoelectron photomicrograph showing abundant reaction product on the cell surface of the labyrinth structure of follicular dendritic cell. x 3700.
INTRATHYROIDAL
LYMPH
FOLLICLE
125
FIG. 5. Immunoperoxidase staining for C3b receptor in the tissue surrounding an adenoma nodule. Electron photograph showing positive staining on the cell surface of a follicular dendritic cell. x 7500.
endogenous Tg-anti-Tg IC. If it is assumed that ICs play an important role in the appearance of GCs, Tg-anti-Tg IC should be more important than the other antibodies (TSH, T,, and T4). Many C receptors on the cell surface have been reported, i.e., Clq, C3a, C3b, iC3b, C3d, C5, and PIH globulin receptors (28). Among these receptors, C3bR and C3d receptor (C3dR) have been considered to play the most important role in the binding of IC. The presence of C3bR on the cell surface of human erythrocytes, monocyte/macrophages, B cells, and certain T cells has been accepted (18, 29, 30). Furthermore, it is also accepted that C3dR is present on the cell surface of human monocyte/macrophages and B cells (30,3 1). Recently, Gerdes and Stein (32) reported that there are many C3 receptors on the DRC in the GCs, and more C3 receptors were found on the DRCs than on the circumscribed B cells. In the present study many GCs were stained in a lacy dendritic pattern with the antisera against Clq, C3c, C3d, C3b INA, and C3bR, and some were also detected on the cell surface of germinocytes, germinoblasts, or FDCs and in the intercellular space within the GCs. Using immunohistochemical methods we recently identified the presence of C3dR on the cell surface in GCs (33). ICs bind to the cell
126
FI(s. 6. Immunosti photl ograph showing tion. indirect immune
KASAJIMA.
YAMAkAU.I\.
4%11) lM.4)
Light micros :opic fixed cryostat sec-
surface by the binding between C3d and C3dK preventing acll\,ation of C5. and then IC binding to C3 is retained on the cell surface of FIX for a long period under more stable conditions. As Klaus rr rrl. (341 mentioned concerning the retention of ICs and presenting of antigens to germinocytex. FIX may play an important role in the immune response within the CJC‘S. On the other hand, late components (C6. C8. and 0) and alternative pathway components (C3 activator and properdin) were not detected within the GCs. It is suggested, however, that late C components may participate under conditions involving the solubilization of ICs (35-37). In the present study we investigated the distribution of T-cell subsets within the GCs of thyroid tissues. Poppema et rrl. (38) reported T-cell subsets within the GCs of human lymph nodes, but there have been no reports of T-cell subsets within the GCs in thyroid tissues. Our results resemble those of Poppema. The number of Leu 3a and OKT 4-positive helper/inducer cells was more than that of Leu 2a and OKT 8 suppressor/cytotoxic cells, and sometimes a group of these positive cells was seen at the periphery of GCs. It is reasonable to assume that T helper inducer cells play an important role in the formation of GCs and in the
INTRATHYROIDAL
127
LYMPH FOLLICLE
TABLE 3 REACTIVITYTOVARIOUSANTISERAWITHINTHEGERMINALCENTERSIN~~CASESWITH SECONDARYLYMPH FOLLICLES Antisera Immunoglobulins 1s kM Id K free chain A free chain Thyroid associated Thyroglobulin Thyroxine Triiodothyronine Thyrotropin Thyroxine-binding globulin Monoclonal antisera Dendritic reticulum cell 1 C3b receptor Leu l,OKT3 Leu 2a. OKT 8 Leu 3a, OKT 4 OKT6
Reactivity
++ +++ + +++ +++ +++ -
+++ +++ + + +
Antisera Complement components Ch c4 c3 c3c C3d C5 C6 C8 c9 C3 activator Properdin Regulatory Protein C3b inactivator BlH globulin
Reactivity ++ -I+ + +++ + -
+ -
Note. + + + , Strongly positive; + + , moderately positive; + . weakly positive; - , negative. These 76 cases comprised 28 with cancer, 23 with chronic thyroiditis, 12 with nontoxic goiter, 8 with adenoma. and 5 with Graves’ disease.
induction of B-cell differentiation within the intrathyroidal secondary LFs as within the other nodal secondary LFs. Moreover, it seems that T cells, B cells, tingible body macrophages, and FDCs are all required for immune reaction within the GCs. ACKNOWLEDGMENTS This work was supported by Grant-in-Aid for General Research (B) No. 59480142, from the Ministry of Education, Science and Culture, Japan. The authors thank Professor Akira Kajita and Professor Mizu Kojima, Tokyo Women’s Medical College, for helpful advice and discussion and also Associate Professor J. Petrich Barron, St. Marianna University School of Medicine. for his revision of the manuscript.
REFERENCES 1. Imai, Y., Terashima, K., Matsuda, M., Maeda, K., and Kasajima, T., Recent Adv. RES Res. 21, 51, 1983. 2. Meissner, W. A., In “The Thyroid: A Fundamental and Clinical Test” (S. C. Werner, and S. H. Ingbar, Eds.), pp. 444-479, Harper & Row, New York, 1978. 3. Clagett, J. A., Wilson, C. B., and Weigle, W. A., J. Exp. Med. 140, 1439, 1974. 4. Kalderon, A. E., and Bogaars, H. A., Amer. J. Med. 63, 729, 1977. 5. Fujiwara, H., Torisu. M., Koitabashi, Y., Baba. T., and Esaki. H., C&n. Immunol. Immunopathol. 19, 98, 1981.
IX
KASAJIMA.
1 ,\MAK,411
t3.
.\hl.i
IMhi
6. Fujiwara. H.. lb1-1>11. 34.. Sugisaki. ‘I’.. anti Ohant). ii ~ i !i; fiJi,,li,,ia ,I !,:,/,~,!/i,~;?,!!i!i’: IY. i<“i 19x1. 7. Yagi, Y.. .-lc’t
July 8. 1986: accepted
with
revision
January
6. 19X7