Immunological features of endemic goiter

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

Immunological

41, 265-272 (1986)

Features

of Endemic

Goiter

A. COSTA,* V. BENEDETTO,~ C. RICCI,§ P. BORELLI,# E. FADDA,$ N. RAVARINO,$ B. TORCHIOA U. DAR~o,$ P. FRAGAPANE,~ G. VARVELLO,? AND V. DE FILIPPIS* *Endocrine Division, +Second General Surgery Division, #Prrthology Itz.rtitrrte Mr:rrri;iw~o und #Depurtment of Clinical Imrn~molog~. CJni,~ersity of Turin. Itcc!\

Hospitrrl,

Some immune aspects of simple endemic goiter have been studied through a comparison of IgG, IgA, IgM. k and A chains, and C3 and C4 in the peripheral blood of 59 patients operated on for goiter and the peripheral blood of 49 normal controls. The median IgM was lower in the goiter blood. The incidence of thyroglobulin (Tg) and microsomal (Mi) antibodies (Abs) was 20.3% in goiter blood and that of nonthyroid autoAbs was 37%. Active and total rosetted blood lymphocytes were counted and OKT3. OKT4. OKT8. Leu 1. Leu3a. LeuZb. T DR + , and NK cell populations were classified. Helper T cells were occasionally decreased when goiter was associated with lymphocytic thyroiditis. The NK percentage was sometimes higher in goiter blood, whereas the T DR+ percentage was not significantly different in the two groups. Lymphocyte infiltration (LI) was noted in 32% of goiters (about 5% with a diffuse and nodular pattern). A prevalence of helper/inducer cells was observed among the infiltrating T cells. HLA-DR antigen (Ag) positive epithelial cells were seen, not only in LI areas. Granular deposits of IgG, IgA, IgM, and C3 on the follicular basal membrane were stained in 6.7% of goiters Patterns histologically and immunologically similar to those in Hashimoto’s thyroiditis may therefore be observed in long-standing simple endemic goiter, suggesting that an autoimmune mechanism may be involved in its pathogenesis. Q 1986 Academic PKS. IX.

INTRODUCTION

The involvement of immune processes in simple goiter has been described since 1960(l). The recent tendency is to regard these manifestations not as occasional epiphenomena of individual cases, but as the expression of an immunologic mechanism involved in thyroid diseases in general. This paper seeks to throw light on the boundaries between immune and nonimmune thyroid diseases. MATERIAL

AND

METHODS

Subjects. Forty-eight women and 11 men aged 45 * 12 years operated for goiter, and 49 normal controls matched for age and sex were compared. The patients were from Piedmont’s endemic goiter areas, where the mean daily iodine urinary elimination is 70 pg in towns and 50 kg in the country. Clinical and histologic investigations showed that these were all normally functioning multinodular goiters, not under the action of drugs. The number of subjects per examination is indicated. Techniques. IgG, IgA, IgM, k, A, C3, C4 values were determined by radial immunodiffusion (2). Thyroid thyroglobulin antibodies (Tg Ab) and microsomal antibodies (Mi Ab) were determined with the TRC technique, using Welcome 265 0090- I 229186 $1.50 CopyrIght ID 1986 by Academx Press. Inc. All rights of reproduction in any form reserved.

266

COSTA

ET AL.

kits. Nonthyroid Abs were measured by indirect IF on cryostatic sections of liver for nuclear Ab (Nu Ab), kidney for Mi Ab, stomach for gastric (Ga) and smooth muscle (Sm) Abs. Active (EA) and total (ET) lymphatic cells were isolated as described elsewhere (3). T lymphocytes and their subclasses were identitied with commercially available MO Abs (OK series, Orto Diagnostic System; Leu series, Becton-Dickinson) and enumerated by eye. T DR+ lymphocytes were also counted in T cells after adherent cell depletion. Nonrosetted mononuclear cells (P M C) were characterized with Leu2b and Leu7 MO Abs (NK). Thyroid lymphocyte infiltration (LI) was evaluated according to the arbitrary grades proposed by Buckingam et al. (4): + indicates a fairly numerous LI with slight alteration of the thyroid parenchyma, + + + indicates diffuse and nodular infiltration with alteration and substitution of wide areas of the thyroid and pronounced oxyphilia. Intrathyroid lymphocytes were immunohistochemically characterized in four thyroids with the epithelial cells expressing HLA-DR Ag. Exams were carried out in areas of various density LI. Using the immunoperoxidase staining method, MO Abs anti-Leul (pan T), anti-Leu3a (T helper/inducer), anti-Leu2b (T cytotoxic/ suppressor), anti-Leu7 (NK), anti-HLA-DR (B lymphocytes and activated T cells) were employed. Immunohistologic examinations were conducted on serial cryostatic sections by direct and indirect IF(5), and Dako anti-human Ig and antiC3 and anti-C4 sera. Statistics. Statistical analysis of the results was performed with Student’s t test. Differences were considered significant when P < 0.05. Results are given as arithmetic means + 1 SEM. RESULTS

Each patient is assigned a single number for the easy location of his or her data in each table. ZgG, ZgA, ZgM, k, X, C3, C4. Values for both groups and the significance of their differences are listed in Table I. Only IgM median proved lower ( - 2 1%) in goiter; individual IgM values were lower than the controls’ mean in 63%, higher in 17%. Thyroid specific Abs were observed in 20.3% of the goiter patients; Tg Ab (l/20, l/5120) in 13%, and Mi Ab (l/100, l/25.000) in 15%. Both Abs were present in 8.5% (Table 2). Nonthyroid autoAbs were observed in 37%: Mi Abs 6.7%, Nu Abs 16.9%, Ga Abs 8.5%, Sm Abs 20% (Table 2). T3 Abs were also present in one patient. Peripheral blood lymphocyte typing and count (Table 3). No significant difference was noted between normal and goitrous subjects in the mean values of T cells (as % of total lymphocytes) nor in the percentage of subsets, irrespective of whether NK or Leu MO Abs were used. The percentage of NK cells was somewhat higher in goiter. The percentage of T DR+ cells was not significantly different in the two groups. LI was present in 19 goiters (32%) in three patients (Patients 5,6,48) in a diffuse form with pale center confluent nodes and plasma cells. About 50% of the patients with LI had Abs in their blood, but in one subject LI was + + + and there

IMMUNOLOGICAL

FEATURES

OF ENDEMIC

267

GOITER

TABLE 1 SERUM I~G,I~A,I~M,L~GHTCHAINS.C~,ANDC~ Normals (49) (mean t SD)

Goitrous (59) (mean -C SD)

IgG (mg/dl)

1365 ? 302

1gA (mgidl)

268 in 110

IgM fmgidll

207 2 71

k&J/ml)

102 ? 29

W/ml)

126 2 41

1264 2 304 (n.s.) 251.4 2 126 (ns.) 163.3 2 51 (P < 0.01) 97.7 2 34 (n.s.) 116.3 k 31 (n.s.) 0.84 2 0.18 (as.) 105.8 t 21 (n.s.) 44.8 + 12.4 (Ils.)

k/h

0.81 t 0.12

C3(mg/dl)

116.4 2 29

C4( mgidl)

45.6 t 13.8

were no circulating Abs. LI was observed in 77% of patients with circulating thyroid Abs. Characterization of the mononuclear cells in the thyroid tissue showed in four patients (5,6,48,57) (Table 4) a prevalence of T cells among the infiltrating lymphocytes. Lymphatic follicles with the germinal center and the neighboring infiltrated areas were stained by the HLA-DR MO Ab. Around the follicles cells TABLE

2

Thyroid Abs in blood (number in parentheses)” Tg Ab (13%): 5(320), 7(5120),18(20). 20(40), 24(40). 26(320), 48(320), 58(40) Mi Ab (15%): 5(400), 6(1600), lO(lOO), 15(25.000), 18(400), 38(400), 46(1600), 48(1600), 57(4000) Nonthyroid auto Abs Microsomal Ab: 5, 6, 15, 38 Gastric Ab: 5, 15, 23, 25, 31 Nuclear Ab: 4, 5, 6, 9, 17, 21, 24. 33, 49, 50 S. muscle Ab: 5, 10, 11, 12, 13, 15, 17, 31, 36, 40, 50, 52 LI of the thyroid6 435, 6. 12, 13. 15, 18,20, 22, 23, 24, 25, 28,29, 39, 42,46, 48, 57 Immunohistologyc 4,5, 6, 8, 15, 28, 29, 31, 38, 48, 57 HLA-DR + thyrocytesd 5, 6, 48, 57 Note. Each patient has been assigned a single number for convenience a Patients with both Tg Ab and Mi Ab italicized. b Patients with LI+ + and LI+ + + italicized. c Cases with immune complexes at the FBM italicized. d Patients with HLA-DR + thyrocytes.

COSTA ET AL.

268 TOTALTCELLSANDSUBSETS.T

TABLE 3 HLA-DR+ CELLSANDNK

CELLSIN~ERIPHERAL

BLOOD

Normals (mean 2 SD)

Goitrous (mean 2 SD)

EA+

55.2 2 6.2

ET+

73.8 5 5.9

OKT3 +

93.0 t 10.0

OKT4+

50.0 k 10.0

OKTS +

36.0 2 8.0

OKT4 + IOKT8 +

1.45 + 0.5

Leul+

77.3 t 11.0

Leu3 +

57.0 2 12.0

Leu2 +

36.0 5 8.0

Leu3 + /Leu2 +

1.64 t- 0.48

NK:Leu7 + ,LeuZ +

2.95 k 3.5

NK:Leu7 + ,LeuZ -

3.20 t 2.34

T HLA-DR +

4.69 t 1.75

51.7 i 1.0 (n.s.) 73.5 rt 10.0 (ns.) 92.5 k 11.0 (n.s.1 45.0 5 16.0 (n.s.) 42.0 2 II.0 (n.s.1 1.18 k 0.6 (n.s.) 78.66 2 12.0 (n.s.) 63.0 i 16.0 (n.s.) 41.6 k 14.0 (n.s.) 1.86 2. 1.0 (n.s.) 3.09 2 4.25 (n.s.) 4.36 +- 2.69 (P < 0.01) 4.56 +- 3.2 (n.s.)

Note. EA + , ET+ cells as % of lymphocytes. OKT+ cells. Leu + cells, T HLA-DR + cells as % of ET + cells. NK cells as % of peripheral mononuclear cells.

stained by the anti-Leul and anti-Leu4 MO Abs predominated. The Leu3a + cells exceeded the Leu2b + . Leu3a + and Leu2b + cells taken together were less frequent than the Leul + cells. Among the follicles both Leu3a+ and Leu2b + cells were not stained with clear-cut prevalence. Leu7+ cells were scattered among the follicles, in both the lymphatic nodes and the germinal centers. HLA-DR + epithelial cells were more easily recognized in the follicles of less densely infiltrated areas. In some follicles these cells were scanty, in others nearly all were of this type. Immunolzistologic examination. Ig were observed in the follicles of 19% of goiters (usually IgG, 2 IgA, 1 IgM). In four IgG and C3 were responsible for granular deposits in the basal membrane of the follicles (patients 5,6,48,57). DISCUSSION

The low IgM observed in a minority (28%) of goiter patients by Mota et al. (6) and (in toxic goiter only) by Kiy et al. (7) has not been explained. In addition,

5

Endemic, familial goiter. Scintiscamlarge, multinodular goiteruneven distribution of the tracer:cold areas. T4 9 pgidl; T3 I30 ngi dl: TSH 0.9 FLU/ml

Endemic, familial. multinodular goiter recurrent after surgery. T4 I I pg/dl: T3 300 ng/dl: TSH 0.8 kU/ml

Endemic, familial multinodular goiter recurrent after surgery. T4 I I pg/dl: T3 220 ng/dl: TSH 1.05 nU/ml

No. 48 C.F., female, 58 years

No. 57 G.M., female, 63 years

Macrofollicular goiter. Diffuse infiltration with lymphocytes and plasma cells. Many lymphoid follicles. IgG, C3 along the FBM and in the stroma. Epithelial cells expressing DR Ag were present. Leu3 + lymphocytes were predominant among infiltrating mononuclear cells. Micro-macrofollicular goiter. Intense, diffuse and nodular LI. Widespread oxyphilia. Many DR+ epithelial cells. The majority of the infiltrating lymphocytes were Leu3 +. Granular staining of IgG and C3 at the FBM. Nodular goiter. Many lymphocytic aggregate and lymphoid follicles. Granular deposition of IgG and C3 along the FBM.DR+ epithelial cells and predominant lymphocyte subpopulation among infiltrating mononuclear cells as in case 5. Nodular colloid cysted goiter. Calcic specks. Moderate. sparse LI. IF and immunohistochemic staining of sections gave similar results.

Endemic, familial, multinodulat goiter. Scintiscan: enlarged goiter: in the right lobe hot noninhibiting adenoma. T4 7.6 pg/dl; T3 224 ng/dl: TSH 1. I I*U/ml

TABLE 4 AND a.i. LYMPHOCYTIC

Pathology

GOITER

Clinical data

No. 6 D.A., female, 48 years

A.B., female. 63 years

No.

Patient

ENDEMIC

600 309 111 59 61 100 55

:

70

IgG : 1370 IgA: 393 IgM: 271 k : 113 I45 :3 i III c4 : 43

A : 83 c3 : 58 c4: 31

k

IgG : 1078 IgA: 85 IgM: 174

IgG: IgA: 1gM: k : A : c3 : c4 :

lg. c3. c4 (mg/dl) k. A (U/ml)

THYROIDITIS

MiAb: l/4000

Tg Ab:

Mi Ab: l/I600

Tg Ab: 11320

MiAb: 111600

Tg Ab:

Mi Ab: 11400

Tg Ab: 11320

Tg Ab Mi Ab

Mi: Ga: Nu: Sm:

Mi: Ga: Nu: Sm:

+ + -

+ + -

Mi: + Ga: ++ Nu: + Sm: +

Nonthyroid autoAbs

EA ET Leul Leu3 Leu? Leu3 Leu2

EA ET Leu I Leu3 Leu2 Leu3 Leu2

: : : : : :

: : : : : :

60% 75% 80% 68% 30% 2.26

59% 66% 61% 16% 58% 0.27

EA : 59% ET : 77% OKT3: 83% OKT4: 35% OKTI: 39% OKT4: 0.89 OKT8

EA : 37% ET : 54% OKT3: 100% OKT4: 44% OKTS: 38% OKT4: I.15 OKTS

T cells and subsets

270

COSTA

ET AL.

Fujiwara et al. (8) report a normal C’ activity in adenomatous goiter. The equal k/X ratio in the two groups is in agreement with the data that Tg Ab and Mi Ab are both of k and A types (9). The increased incidence of thyroid Abs (Tg Ab, Mi Ab) described in most goiter endemics (Ref. (10)) is regarded as a secondary a.i. process. Our highest titers were l/5120 for Tg Ab and l/25.000 for Mi Ab (Table 2). Titers above l/2.000 have been considered evidence for either Hashimoto’s or Graves’ disease (1 l), though differentiation between primary and secondary a.i. processes on the basis of Abs titers is not in agreement with clinical experience. As for the nonthyroid auto Abs, the 8.5% incidence of Ga Abs measured in our patients is clearly below that considered for a.i. thyroiditis and similar to that detectable in elderly normal women (12). The EA, ET cell percentage in multinodular goiter is regarded as normal (14, 15). Our mean OKT3, Leu 1, OKT4, Leu3a values in both groups were within the normal range (16- 18). A low helper/ inducer percentage was only noted in three patients with goiter and intense lymphocytic thyroiditis (Table 4): 16, 29, and 35%, respectively. In Hashimoto’s thyroiditis this percentage is described as normal or decreased (18, 19). OKT,, Leu2b mean percentage values were normal in both groups (20). Diminished cytotoxic/suppressor cell values have been observed in autonomous toxic adenomas (21) and in some cases of euthyroid multinodular goiter (22, 23). Futhermore the mean percentage of both NK cells and T DR+ lymphocytes is normal in goiter patients (24-26). Percentage and activity of peripheral NK are normal in a.i. thyroiditis (27), whereas the frequency of activated T cells is increased (28). On the whole, our data seem to confirm that the peripheral lymphocyte subset percentages are unchanged in chronic thyroid disorders (29). The LI of the gland in some of our patients (Table 4) was diffuse, with lymphoid germinal centers, plasma cells, and epithelial eosinophilia. Hyperthyroid adenomas and also euthyroid goiters in which the volume of the LI, the plasma cell density, and the percentage of Ig-producing plasma cells were the same as in Graves’ disease and in Hashimoto’s thyroiditis have been reported (30). In four patients the immunohistologic examination showed IgG and C3 immunocomplexes at the follicular basal membrane (FBM): this has been reported in Graves’ disease and in Hashimoto’s thyroiditis (8) (31) but not for simple goiter. Immunohistochemic characterization of the mononuclear cells infiltrating the goiter showed the prevalence of T cells, mainly of the helper phenotype, in accordance with the few data provided for simple goiter (32, 33). Ia + thyrocytes in nodular goiter have also been observed by Jansson et al. (34) and Leclerc et al. (35), and in autonomous hyperfunctioning adenomas by Grubeck-Loebenstein et al. (36) and by Orlandini et al. (37). Epithelial cells expressing class II MHC Ags may promote or perpetuate an autoimmune cycle (38). Our data show that in simple goiter signs of serological and cell-mediated immunity may appear; i.e., in about 50% of our subjects operated upon. Since such markers are rare in the early stages of the endemic goiter (39, 40) we may draw the conclusion that it is not of autoimmune origin. The a.i. process is more frequent in middle aged women, in goiters recurrent after surgery, or when autonomous tissue or toxic adenomas are involved. Subsequent lymphocytic thyroiditis (focal and presumably superimposed) may differ from the Hashimoto’s thy-

IMMUNOLOGICAL

FEATURES

OF ENDEMIC

GOITER

271

roiditis. In 5% of the operated goiters, however, no clear-cut distinction could be drawn between the two forms in the light of our morphologic and immunologic data. In conclusion, both the previous work (IO) and our data point at a possible overlapping between real autoimmune thyroid disorders and other pathological thyroid conditions in which immunologic mechanisms are involved, although probably secondarily. ACKNOWLEDGMENT We thank Mrs. W. Yansen for excellent technical assistance.

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