AUTOIMMUNE THYROID DISORDERS - AN UPDATE - 2005.pdf - Pb Med - ao87

Indian Journal of Clinical Biochemistry, 2005, 20 (1) 9-17

AUTOIMMUNE THYROID DISORDERS - AN UPDATE

Manorama Swain*, Truptirekha Swain** and Binoy Kumar Mohanty***

Department of Biochemistry*, Pharmacology**, Endocrinology***, M.K.C. G Medical College, Berhampur and

S.C.B. Medical College, Cuttack**

ABSTRACT

Background : Autoimmune thyroid disease (AITD), a common organ specific autoimmune

disorder is seen mostly in women between 30-50 yrs of age.Thyroid autoimmunity can cause

several forms of thyroiditis ranging from hypothyroidism (Hashimoto's thyroiditis) to

hyperthyroidism (Graves'Disease). Prevalence rate of autoimmune mediated hypothyroidism is

about 0.8 per 100 and 95% among them are women. Graves' disease is about one tenth as

common as hypothyroidism and tends to occur more in younger individuals. Both these disorders

share many immunologic features and the disease may progress from one state to other as the

autoimmune process changes. Genetic, environmental and endogenous factors are responsible

for initiation of thyroid autoimmunity. At present the only confirmed genetic factor lies in HLA

complex (HLA DR-3) and the T cell regulatory gen~ (CTLA 4). A number of environmental factors

like viral infection, smoking, stress & iodine intake are associated with the disease progression.

The development of antibodies to thyroid peroxidase (TPO) thyroglobulin (TG) and Thyroid

stimulating hormone receptor (TSH R) is the main hallmark of AITD. Circulating T Lymphocytes

are increased in AITD and thyroid gland is infiltrated with CD4+ and CD8+ T Cells. Wide varieties

of cytokines are produced by infiltrated immune cells, which mediate cytotoxicity leading to

thyroid cell destruction, Circulating antibodies to TPO and TG are measured by

immunofluorescense, hemagglutJnation, ELISA & RIA. TSHR antibodies of Graves' disease can

be measured in bioassays or indirectly in assays that detect antibody binding to the receptor.

KEYWORDS

Autoimmune thyroid disease, thyroid peroxidase antibodies, Thyroglobulin antibodies, and TSHR

antibodies

as hypothyroidism affecting mostly the younger

individuals (2). Many of these patients progress to

hypothyroidism either spontaneously after treatment

with antithyroid drugs or iatrogenicallyafter radioiodine

therapy or surgery. The development of antibodies to

thyroid peroxJdase(TPO), thyroglobulin(TG) and thyroid

stimulating hormone receptor (TSH-R) is the main

hallmark of AITD (3).

INTRODUCTION

Autoimmune thyroid disease (AITD) is a common

organ specific autoimmune disorder affecting mostly

the middle aged women. About 2 to 4 percent of women

and up to 1% of men are affected worldwide, and the

prevalence rate increaseswith advancing age (1). AITD

comprises a sedes of interrelated conditions including

hyperthyroid Graves' disease (GD), Hashimoto's

(goitrous) thyroiditis, atrophic autoimmune

hypothyroidism, postpartum thyroiditis (PPT) and

thyroid associated orbitopathy (TAO) .Out of all these

diseases, Hashimoto's thyroiditis (HT) and Graves'

disease (GD) are the commonest type and share many

features immunologically. One form of the disease may

change to other as the course of the immune process

progresses. Autoimmune hypothyroidism (AH) affects

about 5 to 10% of middle aged and elderly women.

Graves' disease (GD) is about one tenth as common

ETIOLOGY

The etiology of AITD is multifactorial. Susceptibility to

the disease is determined by a combination of genetic,

environmental and constitutional factors.

GeneUcFactors:

Numerous studies show a higher frequency of AITD in

family members of patients with autoimmune

hypothyroidism and Graves' disease (4). Both types of

the disease cluster together in families, provides

additional support that these conditions share

common etiologic and pathogenic features. The

autoimmune polyglandular syndrome type 2, involves

the occurrence of autoimmune thyroid dysfunction with

other autoimmune diseases. (Type 1 diabetes

Author for correspondence

Manorama Swain

Department of Biochemistry

M.K.C.G Medical College, Berhampur - 760 004

Indian Journal of Clinical Biochemistry, 2005

Indian Journal of Clinical Biochemistry, 2005, 20 (1) 9-17

Chinese Graves' disease (GD) patients, was

associated with the relapse of the hyperthyroidismafter

antithyroid withdrawal. NG polymorphism of the

cytotoxic T lymphocyte-associated molecule-4 gene

affects the progress of GD. The GIG genotype is

associated with poor outcome (13).

mellitus, Addison's disease, pernicious anemia &

vitiligo). Shared genetic factors are likely in this group

of autoimmune disorders.

Twin studies show increased concordance of GD and

AH in monozygotic (MZ) twins, compared with dizygotic

(DZ) twins. A recent invsstigationbased on a population

of 8,966 Danish twins has shown that concordance

for GD in MZ twins was 35% compared with 3% in DZ

twins (5). Various techniques have been employed to

identify the genes contributing to the etiology of AITD,

including candidate gene analysis and whole genome

screening. These studies have enabled the

identification of several loci (genetic regions) that are

linked with AITD, and in some of these loci putative

AITD susceptibility genes have been identified. Some

of these genes/loci are unique to Graves' disease (GD)

and Hashimoto's thyroiditis (HT) and some are

common to both diseases, indicating that there is a

shared genetic susceptibility to GD and HT. The

putative GD and HT susceptibility genes include both

immune modifying genes (e.g. HLA, CTLA-4) and

thyroid specific genes (e.g. TSHR, TG). Most likelythese

loci interact and their interactions may influence

disease phenotype and severity (6).

Environmental Factors:

Iodine: It has been well documented that the incidence

of AITD is proportional to dietary iodine content. In

Europe the prevalence of GD increases with national

iodine intake programs. Iodine increases the

antigenicity of TG with exacerbation of experimental

thyroidi~ in animals. Recent in vitro studies in

NOD.H2" mouse have shown that high iodine doses

alone may damage thyrocytes and enhances the

disease progression in a dose-dependent manner

(14).

Infection: No convincing evidence has indicated a role

infection in All except congenital rubella syndrome. An

association has been proved between Yersinia

infection and GD (15). Yersinia contains proteins that

mimic TSH-R immunologically. Recently, retrovirus has

received attention but the results are conflicting (16).

The most important susceptibility factor so far

recognized is association of AITD with HI.A-DR alleles.

These MHC class-II genes play a cdtical role in the

initiation of adaptive immune response. HLA-DR3 is

the best-documented genetic factor for GD and AH in

Caucasians (7). In non-white populations, GD is

associated with different HLA alleles. For example, it

is associated more with HLA B35, B46, A2, and

DPBI*0501 in Japanese; (8)A10, B8, and DQw2 in

Indians; (9) and DR1 and DR3 in South African blacks

(10).

Stress: Some studies suggest an association

between antecedent major life events and Graves'

disease, but a causal role of stress in autoimmune

process remains to be clearly established. Smoking

is a minor risk factor for the development of thyroid

ophthalmopathy (17). The female preponderance of

thyroid autoimmunity is most likely due to the influence

of sex steroids. Estrogen use is associdted with a

lower dsk, and pregnancy with a higher risk for

developing hyperthyroidism (18).

PATHOLOGY

The cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene,

encoding a negative regulator of the T-lymphocyte

immune response, had been reported to be associated

and/or linked to AITD. Recently, AITD susceptibility in

the Caucasians was mapped to the 6.1-KB3'UTR of

the CTLA-4 gene, in which the three single nucleotide

polymorphisms (SNPs) CT60, JO31, and JO30 were

strongly associated with AITD. The SNP, JO31 was

most significantly associated with AITD in the

Japanese, whereas the association of the JO30 with

AITD was not observed (11). Ueda H & coworkers have

identified polymorphism of CTLA-4 gene that is a

candidate gene for common autoimmune disorders

like GD, AH and Type-1 Diabetes ('riD). The diseasl~

susceptibility was mapped to a non-coding 6.1 KB3'

region of CTLA-4, the common allelic variation of which

was correlated with lower mRNA levels of the soluble

alternative splice form of CTLA-4 (12). It was recently

shown, that the A/G single nucleotide polymorphism

(SNP) at position 49 in exon 1 of the cytotoxic T

lymphocyte-associated molecule-4 gene in 148

In Hashimoto's thyroiditis there is an extensive

infiltration of thyroid by lymphocytes, plasma cells and

macrophages. There is formation of germinal center

and giant (Langerhans) cell can occur. The thyroid

follicular cells are destroyed to a variable extent,

depending on the chronicity of the disease. Dudng

this process the remaining cell become hyperplastic

and undergo oxyphilic metaplasia, which gives rise to

the so-called Askanazy or Hurthle cells.

The pathologic features of Graves' disease are often

obscured by priortreatment with antihyroiddrugs. There

is hypertrophy and hyperplasia of the thyroid follicles,

the epithelium is columnar and the colloid s:hdnks. In

addition a variable degree of lymphocytic infiltration is

present, sometimes with germinal center fom~ation.

Autolmmune features:

All forms of thyroid autoimmunity are associated with

Indian Journal of Clinical Biochemistry, 2005

Indian Journal of Clinical Biochemistry, 2005, 20 (1) 9-17

a lymhocytic infiltrate in the thyroid, and these

lymphocytes are largely responsible for generating

both T and B Cell-mediated autoreactivity. Other sites

such as thyroid draining lymph nodes and bone marrow

may also contain thyroid autoreactive lymphocytes in

AITD. The initial autoimmune response by CD4+ T

cells appears to up regulate the secretion of IFN-g

resulting in the enhanced expression of MHC class II

molecules on thyrocytes. This most likely triggers

expansion of autoreactive T cells and gives rise to the

characteristic inflammatory response and as the

disease progresses; thyrocytes are targeted for

apoptosis resulting in hypothyroidism. Another

contributing factor to the observed hypothyroidism in

Hashimoto's thyroiditis patients could be the circulating

TSH inhibitory antibodies. Graves' disease on the other

hand represents the other end of spectrum wherein

the patients suffer from hyperthyroidism. The activation

of thyroid specific CD4+ T cells leads to the recruitment

of autoreactive B cells and the mounting of thyroid

stimulatory immune response via anti-thyroid

antibodies (19).

out that poorly iodinated TG is only weakly

immunogenic (25).

Thyroglobulin autoantibodies are found in less than

60% of patients with lymphocytic thyroiditis and 30% of

Graves' disease patients. They are polyclonal and

mainly of IgG class with all four subclasses

represented. TSH regulates the cell surface

expression of TPO and TG altering the mRNA

transcription of these two proteins, possibly at the gene

promoter level. These effects are mimicked by auto

antibodies (both blocking and stimulating) in the sera

of the patients with GD (26).

iii. Thyroid Stimulating Hormone receptor (TSH-R)

Antibodies

TSH-R is the prime autoantigen in Graves' disease

and atrophic thyroiditis. It is located on the basal

surface of thyroid follicular cells. In Graves' disease

thyroid stimulating antibodies (TSAbs) bind to the

receptor and stimulate the thyroid cell to produce

excessive amount of thyroid hormones resulting in

hyperthyroidism. In patients with atrophic thyroidiUsthe

major antibody is the TSH-R blocking antibody. After

binding to the receptor this antibody blocks the binding

of TSH to its receptor, thus preventing stimulation of

thyroid cell. This results in diminished thyroid hormone

output, atrophy of thyroid gland and the clinical state of

hypothyroidism.

Autoantibodies:

i. Thyroid Peroxidase (TPO) antibodies:

TPO is the key thyroid enzyme catalyzing both the

iodination and coupling reaction for the synthesis of

thyroid hormone. It is membrane bound and found in

the cytoplasm and in high concentration on the apical

microvillar surface of thyrocytes. It is of mol wt between

100 to 105-kDa and previously was known as thyroid

microsomal antigen (20). Multiple T & B Cell epitopes

exists within the molecule and the antibody response

to TPO is restricted at the level of the germ line heavy

and light chain variable (V) region (21).

TSH-R has 398 amino acid extra cellular domains, a

266 amino acid transmembrane domain (organized

in seven loops) and an 83 amino acid intracellular

domain. Antibodies binding to the amino terminal area

are stimulatory where as those binding to amino acids

261 to 370 or 388 to 403, near the cell surface, have

blocking activity (27). As with TPO multiple T and B cell

epitopes has been defined within the molecule.

Anti-TPO autoantibodies are found in over 90% of

patients with autoimmune hypothyroidism and Graves'

disease. Together with TG antibodies these are the

predominant antibodies in AH. Anti-TPO antibodies are

mainly of the IgG class with IgG 1 and IgG4 subclasses

in excess (22).

iv. Other antibodies

Na+ / I" symporter (NIS) is the fourth major thyroid

autoantigen; first demonstrated using cultured dog

thyroid cells. Around a third of Graves' disease sera

and 15% of Hashimoto's sera contain antibodies that

inhibit NIS mediated iodide uptake in vitro (28).

Antibodies to thyroid hormone can be found in 10% to

25% of patients with AITD and non-specific

autoantibodies against DNA, tubulin and other

cytoskeletal proteins can also be detected in a small

proportion of patients.

ii. Thyroglobulin (TG) Antibodies

TG is a 660-kDa glycoprotein composed of two identical

subunits of 330 kDa each. It is secreted by the thyroid

follicular cells into the follicular lumen and stored as

colloid. Each TG molecule has around 100 tyrosine

residues, a quarter of which are iodinated. These

residues couple to form the thyroid hormones

tdiodothyronine (T3) and thyroxin (T4). The sequence

of human TG has-been determined (23). When TSH

stimulates the thyroid cell, TG is endocytosed and

hydrolyzed in lysosome releasing T3 & T4.The exact

location of T and B cell epitopes withi'n TGis uncertain

(24). A key T cell epitope in the spontaneous thyreiditis

of OS chickens contains iodine, and it has been found

MECHANISM OF THYROID CELL INJURY

Several antibody and cell-mediated mechanisms

contribute to thyroid injury in autoimmune

hypothyroidism. Different groups have analyzed

partners in the diseased thyroid glands. In general, in

case of Hashimoto's thyroiditis, the expressions of

death receptors such as CD95 and death receptor

Indian Journal of Cfinical Biochemistry, 2005

Indian Journal of Clinical Biochemistry, 2005, 20 (1) 9-17

ligands such as CD95L and TRAIL in the thyroid tissue

appear to be much higher compared to their normal

counterparts. Also, the expression of positive effectors

of apoptosis such as caspase 3 and 8, as well as Bax

and Bak appear to be relatively high in thyroiditis

samples as compared to controls. This expression

pattern clearly supports enhanced apoptosis as the

mechanism underlying the loss of thyrocytes in

Hashimoto's thyroiditis. In contrast, the opposite trend

appears to be the norm in Graves' disease. One stdking

feature is the highly elevated expression of negative

modulators of apoptosis such as cFLIP, Bcl-2 and Bcl-

XL and close to normal expression of the caspases in

thyrocytes analyzed from Graves' patients, clearly

supporting a role for apoptosis inhibitory mechanisms

in these patients. Although in both cases there is

significant expression of Fas/CD95 and its ligand, only

in Hashimoto's thyroiditis, the thyrocytes undergo

apoptosis. Recent work by Stassi et al. solvedthe puzzle

by exposing the role of cytokines in the development of

autoimmune disorders (29). In case of Hashimoto's

thyroiditis, a TH1 disease, the cytokine IFN-g appears

to play a crucial role in the pathology of the disease by

enhancing the expression of caspases and there by

sensitizing cells to FAS mediated apoptosis. In contrast

in the TH2 mediated Graves' disease, the cytokines

IL4, and IL-10 strongly up-regulate the expression of

two anti-apoptotic proteins Bcl-XL and cFLIP, which

offer resistance to Fas mediated apoptosis. This again

proves the necessary modulatory roles played by the

TH1 and TH2 cytokines in the development of

autoimmune disorders (19).

T-Cell response

Both CD4+ and CD8+ T-Cells occur in thyroid

lymphocytic infiltrate with a preponderance of CD4+

cells. There is an increase in activated T Cell

expressing markers like HLA-DR. A wide array of

cytokines including IL-2, Interferon (IFN-g), Tumor

necrosis factor (TNF-a), IL-4, IL-6, IL-10, IL-12, IL-13

and IL-15 are produced by the lymphocytes with some

variation between patients (32). Thyroid cells express

MHC class-II molecules as well as other

immunologically important molecules and behaves

as an antigen-presenting cell (APC). Expression of

ICAM-1, LFA-3 and MHC class I molecules by thyrocytes

is enhanced by IL-1, TNF-a & IFN-g (33). This response

increasesthe ability of cytotoxic T cells to mediate lysis.

Thyroid cell destruction is mediated both by perforin

containing Cells which accumulate in the thyroid and

by Fas dependent mechanisms (34,35). Cytokines and

other toxic molecules such as nitdc oxide and reactive

oxygen metabolitas probably also contribute directly to

cell mediated tissue injury. (Fig 1 & Fig 2)

Humoral immunity exacerbates cell-mediated damage

in a secondary fashion, both by direct complement

fixations (TPO antibodies) and by ADCC (36).

Complement attack initiated via the classic or

altemative pathway, impairs the metabolic function of

thyroid cells and induces them to secrete IL-I, IL-6,

reactive oxygen metabolites and prostaglandin. All of

these enhance the autoimmune response.

As well as T and B cell, dendritic cells and monocyte/

macrophages accumulate in the thyroid. Presumably

they play a major role as APC & capable of providing

co-stimulatory signals. Thyroid cell-derived monocyte

chemoattractant-I, produced after TNF-a, IFN-g, or IL-I

stimulation, is likely to be responsible for the

accumulation of monocytes, which are important

source of cytokines (37).

B Cell responses

TG and TPO antibodiesoccur in very high concentration

in patients with Hashimoto's thyroiditis and primary

myxedema. These antibodies are less common, but

still frequent in Graves' disease, where as TPO rather

than TG antibodies are frequent in postpartum

thyroiditis (30). Both of the antibodies show partial

restriction to the IgG1 and IgG4 subclass (21). TG

antibodies usually mediate Antibody mediated

cytotoxicity (ADCC), where as TPO antibodies form

terminal complement complexes within the thyroid

gland. Cell mediated injury may be necessary for TPO

antibodies to gain access to their antigen and become

pathogenic (31).

CLINICAL FEATURES

The main clinical features of hypothyroidism are

summarized in Table-1. Patients with Hashimoto's

thyroiditis may present a goiter which vades from small

to large in size. It is usually firm and painless often

with an irregular bosselated surface. The goiter in case

of GD is diffusely enlarged and firm in consistency.

Increased blood flow may be manifested by a thdll or

bruit. The clinical features of hyperthyroidism are

summarized in Table -2.

Thyroid stimulating antibodies (TSAbs) of Graves'

disease are detected in 95% of cases. These

antibodies are often k chain restricted and of the IgG1

subclass. TS-Abs also occurs in 10% to 20% of

patients with autoimmune hypothyroidism(AH) but their

effects are obscured by TSH-R-blocking antibodies

and destructive processes (27). TSHR is a member of

G protein coupled receptor family. It's stimulation by

TSH leads to intracellular signaling by cyclic

adenosine mono phosphate (cAMP) pathway.

DIAGNOSIS OF AITD

Diagnosis of AITD is based upon clinical features &

supported laboratory investigations. The patient may

be euthyroid, hypothyroid or hyperthyroid, according to

disease type and stage. Investigations used to

determine the existence and cause of both hypo and

Indian Journal of Clinical Biochemistry, 2005

Indian Journal of Clinical Biochemistry, 2005, 20 (1) 9-17

Complemel~//

fixation //~

Expressionof

complement

regulatory proteins

Thyroid

antibody

production

HLA classII molecule

expression

leadingto

//~

anergy of naiveCD4 +

IL-I, TNF,'y-IFN.~

T cells but stimulationof

B cell

differentlaU~

CD4 +T cellsnot needing

costimulation

THYROID

FOLLICULAR CELLS.~

LY~MPHOCYTIC

IL-1,ID 6, IL- 8, 11~

(

/. I ~

12, IL-13,1L-I5

Expressionof HLA

Thl exp~InsldO

class I plusadhesion

molecules

Expressionof Fas and

| ~

jq /

CD8 + cytotoxJc ~

nitric oxide production

T ceils

Destructive ~

thyroiditi~

Fig 1. Interaction between thyroid cells and the immune system via cytokines.

TARGETCELL

CYTOTO~

FasL~

LYMPHOCYTE

Fas

CAPSPASE

ACTIVATION

CELLULARPROTEOLYSIS

DNA FRAGMENTATION

DD : Death Domain proteins

APOPTOSIS

Fig 2. Interaction between Fas ligand (FasL) on cytotoxic lymphocyte and Fas on a target cell leading

to apoptosis

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