Grave Disease

0163-769X/03/$20.
00/0 Endocrine Reviews 24(6):802 835

Printed in U.S.A. Copyright 2003 by The Endocrine Society
doi: 10.1210/er.2002-0020
Current Perspective on the Pathogenesis of Graves

Disease and Ophthalmopathy
BELLUR S. PRABHAKAR, REBECCA S. BAHN, AND TERRY J. SMITH
Department of Microbiology and Immunology (B.S.P.), College of Medicine, University of Illinois at Chicago, Chicago,
Illinois 60612-7344; Division of Endocrinology (R.S.B.), Department of Medicine, Mayo Clinic, Rochester, Minnesota 55901;
Division of Molecular Medicine (T.J.S.), Harbor-UCLA Medical Center, and the David Geffen School of Medicine at
University of California at Los Angeles, Torrance, California 90822; and Long Beach Veterans Administration Healthcare
System (T.J.S.), Long Beach, California 90822
Graves disease (GD) is a very common autoimmune disorder curs in a subset of patients with GD. As in GD, both environ-
of the thyroid in which stimulatory antibodies bind to the mental and genetic factors play important roles in the
thyrotropin receptor and activate glandular function, result- development of GO. Although a number of putative ocular
ing in hyperthyroidism. In addition, some patients with GD autoantigens have been identified, their role in the pathogen-
develop localized manifestations including ophthalmopathy esis of GO awaits confirmation. Extensive analyses of orbital
(GO) and dermopathy. Since the cloning of the receptor cDNA, tissues obtained from patients with GO have provided a
significant progress has been made in understanding the clearer understanding of the roles of T and B cells, cytokines
structure-function relationship of the receptor, which has and chemokines, and various ocular tissues including ocular
been discussed in a number of earlier reviews. In this paper, muscles and fibroblasts. Equally impressive is the progress
we have focused our discussion on studies related to the mo- made in understanding why connective tissues of the orbit
lecular mechanisms of the disease pathogenesis and the de- and the skin in GO are singled out for activation and undergo
velopment of animal models for GD. It has become apparent extensive remodeling. Results to date indicate that fibroblasts
that multiple factors contribute to the etiology of GD, includ- can act as sentinel cells and initiate lymphocyte recruitment
ing host genetic as well as environmental factors. Studies in and tissue remodeling. Moreover, these fibroblasts can be
experimental animals indicate that GD is a slowly progressing readily activated by Ig in the sera of patients with GD, sug-
disease that involves activation and recruitment of thyro- gesting a central role for them in the pathogenesis. Collec-
tropin receptor-specific T and B cells. This activation even- tively, recent studies have led to a better understanding of the
tually results in the production of stimulatory antibodies that pathogenesis of GD and GO and have opened up potential new
can cause hyperthyroidism. Similarly, significant new in- avenues for developing novel treatments for GD and GO. (En-
sights have been gained in our understanding of GO that oc- docrine Reviews 24: 802 835, 2003)
I. Introduction B. Recent studies on the pathogenesis of experimental

II. Risk Factors for Developing Graves Disease (GD) GD
A. Genetic factors VII. Graves Ophthalmopathy
B. Environmental factors A. Clinical features
C. Y. enterocolitica and GD B. Pathological findings
D. Cross-reactivity of Y. enterocolitica proteins with TSHR VIII. Genetic and Environmental Contributions to GO
III. Immunological Basis for GD Pathogenesis
IV. Autoimmune Responses to Thyroid Autoantigens A. Genetic contributions
V. TSH Receptor as an Autoantigen B. Smoking
VI. Evolution of Autoimmune Response to TSHR C. Radioiodine treatment for GD
A. Early studies on pathogenesis of experimental GD IX. Orbital Autoimmunity
A. Immunohistochemical studies of orbital tissues
Abbreviations: APC, Antigen-presenting cell; CRP, cross-reactive B. Orbital T cell repertoire
protein; CTLA4, cytotoxic T lymphocyte antigen-4; DC, dendritic cell;
ECM, extracellular matrix; ETSHR, extracellular domain of human
C. Cytokine production in the orbit
TSHR; GAG, glycosaminoglycan; GD, Graves disease; GO, Graves D. Cytokine effects in the orbit
ophthalmopathy; HAS, hyaluronan synthase; HLA, human leukocyte X. Orbital Autoantigens
antigen; ICAM-1, intercellular adhesion molecule-1; IFN-, interferon-; A. Target cells and candidate autoantigens
IL-6R, IL-6 receptor; LP, lipoproteins; MBP-ECD, maltose binding pro- B. TSHR
tein extracellular domain; MHC, major histocompatibility complex;
mPGES, microsomal PGE2 synthase; NF, nuclear factor; PG, prostaglan- C. Orbital T cell reactivity
din; PGHS, PG endoperoxide H synthase; PPAR-, peroxisome prolif- XI. Role of Orbital Connective Tissue in GO
erator-activated receptor-; RANTES, regulated on activation, normal T A. Orbital connective tissue is targeted by the immune
cells expressed and secreted; TAO, thyroid-associated ophthalmopathy; system in GD
TBII, TSH-binding inhibitor Ig; TCR, T cell receptor; TSAb, thyroid- XII. Role of GAGs in GO
stimulating antibody; TSBAbs, thyroid stimulation blocking antibodies;
TSHR, TSH receptor; TSHR-Abs, TSHR-antibodies; UDP, uridine A. GAGs accumulate in GO and dermopathy
diphosphate; UDP-GD, UDP-glucose dehydrogenase; V, variable; YOP, B. Orbital fat expansion and muscle enlargement can con-
Yersinia outer membrane protein. tribute substantially to tissue remodeling in GO
802
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Prabhakar et al. Graves Disease and Ophthalmopathy Endocrine Reviews, December 2003, 24(6):802 835 803
XIII. Role of Orbital Fibroblasts in GO lies the gender preference noted in GD, as in many other
A. The putative role of orbital fibroblasts in the patho- autoimmune diseases, needs to be elucidated.
genesis of GO
B. Orbital fibroblasts are different from many other
types of fibroblasts A. Genetic factors
C. Orbital fibroblasts express high levels of inducible Increased incidence of GD among members of a family
cyclooxygenase and produce extremely high levels of indicates that genetic factors might play an important role in
PGE2 when activated by proinflammatory cytokines determining susceptibility to GD (1, 2). Studies involving
D. Fibroblasts are sentinel cells capable of initiating lym-
twins indicate a higher degree of concordance, suggesting
phocyte recruitment and tissue remodeling
that the genetic factors might be a major contributor (3).
E. Fibroblasts from the orbit synthesize high levels of
hyaluronan
Earlier studies have shown that patients with GD express
F. Orbital fibroblasts display high levels of CD40, an human leukocyte antigen (HLA)-B8 more often than the con-
important activation molecule trol subjects without the disease (4). Other studies have
G. Do GD-specific IgGs activate fibroblasts? shown that the risk of developing the disease is higher
XIV. Future Perspective among individuals with an MHC (major histocompatibility
complex) class-II haplotype of HLA-DR3 (5, 6) or with
DQA1*0501 haplotype (7, 8). In contrast, the expression of
I. Introduction HLA DR 1*07 appears to confer protection (9). However,
there is racial variation in the association of MHC haplotypes
G RAVES DISEASE (GD) is a well-characterized disease

that is often diagnosed on the basis of clinical im-
pression. Laboratory and pathological findings often provide
with increased risk for the development of GD (9). Two
recent reviews on genetic susceptibility to GD have summa-
rized a large number of studies to date (10, 11). The general
important confirmatory information. GD is thought to rep- conclusions one can draw from studies to date are that mul-
resent an autoimmune process of the thyroid gland in which tiple genetic factors appear to contribute to the risk of de-
stimulatory autoantibodies bind to the TSH receptor (TSHR) veloping GD and the penetrance of the disease is about 30%
and activate gland function leading to hyperthyroidism, of- in monozygotic twins. Some of the susceptibility genes are
ten accompanied by thyromegaly. The disease is accompa- involved in the generation of immune responses and/or
nied by a number of symptoms directly referable to thyroid associated with X chromosome, whereas others appear to be
hormone excess. In addition, some patients with GD develop specific to GD (e.g., GD-1, -2, and -3).
manifestations in localized regions of the connective tissue How could these gene products play a role in protecting
system, including Graves ophthalmopathy (GO) and der- against or enhancing the risk for the development of the
mopathy. It is currently believed that the connective tissue disease? Hyperthyroidism in GD is mediated by autoanti-
manifestations of GD are not a direct consequence of alter- bodies to TSHR protein, and the protein antigens are T cell-
ations in thyroid function but more likely reflect the under- dependent antigens. Therefore, CD4 T cells, most likely,
lying autoimmune processes. Many patients have antibodies play an important role because they provide the necessary
against the TSHR and thyroid peroxidase, and about 50% of help for autoantibody production. If this were true, then the
the patients have antibodies against the thyroglobulin. most effective antigen presentation would be in the context
Pathological findings in the thyroid reflect the hyperthy- of MHC class-II molecules, and thus they may play a critical
roidism associated with GD. A salient feature of thyroid role in the pathogenesis of GD. MHC class-II molecules form
pathology is hypertrophy and hyperplasia of the paren- peptide binding grooves, the specificity of which is deter-
chyma. A change in the thyroid epithelium is a common mined by their amino acid sequence. This allows binding of
finding, with the appearance changing from cuboidal to co- antigenic peptides of a certain length and with specific se-
lumnar with papillary infoldings. In addition, these cells quences. T cells, bearing cognate receptors, in turn recognize
demonstrate enlarged Golgi, increased numbers of mito- these MHC-peptide complexes and undergo activation.
chondria, and vacuolation of the colloid. These changes in the Thus, MHC molecules play a very critical role in the antigen
thyroid are often accompanied by a diffuse cellular infiltra- presentation and thus in the T cell repertoire generation.
tion consisting of lymphocytes and plasma cells. Occasion- Early in the thymocyte development, immature T cells
ally, the infiltration can be more focal and resemble changes migrate from bone marrow into the thymus where they un-
seen in Hashimotos thyroiditis. dergo maturation and selection. Although T cells that react
with self-antigens expressed on the thymic epithelium are
deleted, some self-reactive T cells escape negative selection
II. Risk Factors for Developing Graves Disease (GD) because many tissue-specific antigens may not be expressed
on the thymic epithelium. However, cells capable of inter-
It is well known that most autoimmune diseases are more acting with exogenous antigens and some cells that can in-
prevalent in females than in males. This is true for GD as well, teract with self-antigens are positively selected and migrate
and the disease is six to eight times more common in females to the periphery (12). Because a given MHC molecule can
than in males. The disease most often occurs between 30 and bind only a limited number of peptides with certain se-
50 yr of age, suggesting that yet unidentified age-related quences, they largely determine the repertoire of T cells that
factors and/or hormonal changes may contribute to en- are either eliminated in the thymus (negative selection) or
hanced susceptibility. The molecular mechanism that under- allowed to migrate to the periphery (positive selection). Be-
804 Endocrine Reviews, December 2003, 24(6):802 835 Prabhakar et al. Graves Disease and Ophthalmopathy
cause individuals expressing HLA DR3 and DQA1*0501 hap- signal to antigen-specific T cells, which results in either a
lotypes have an increased risk of developing the disease, it failure to activate T cells or induction of anergy. There are a
is possible that they might fail to present certain tissue- number of potential mechanisms by which an environmental
specific pathogenic peptides in their peptide binding groove agent could trigger an autoimmune response. These include
and thus prevent negative selection of self-reactive T cells induction of an inflammatory response leading to the pro-
that express the cognate T cell receptor (TCR) on their sur- duction of proinflammatory cytokines that can cause en-
face. These self-reactive T cells could encounter the antigen hanced/aberrant expression of MHC class-II and costimu-
in the periphery and undergo activation. In contrast, HLA latory molecules. This could lead to an otherwise innocuous
DR 1*07, which confers protection, might bind pathogenic presentation of self-peptide by MHC molecules into an ef-
peptides and facilitate negative selection of self-reactive T fective antigen presentation and cause activation of antigen-
cells, expressing cognate TCRs, in the thymus and prevent an specific T cells. Thus, cytokine production and/or an imbal-
autoimmune response. Thus, MHC gene products can dif- ance in cytokines caused by infection could lead to the
ferentially affect both the repertoire selection in the thymus initiation of an autoimmune response. The role of cytokines
and subsequent autoimmune responses in the periphery. in GD has been discussed in a recent review (19), and the role
More recently, a number of reports have suggested an of cytokines as it relates to GO will be discussed later. Mi-
association between cytotoxic T lymphocyte antigen-4 crobial infections can also cause overexpression (e.g., heat
(CTLA4) and GD. The CTLA4 protein exists in multiple shock proteins) and/or altered expression of certain self-
isoforms due to genetic polymorphism in the first exon (13). proteins (altered self), which will either provide the neces-
This is often linked with another polymorphism of AT sary strength of signal or be perceived as foreign (20).
dimers at the 3-untranslated region of the third exon, which Many microbial products can act as either B cell or T cell
is thought to be associated with increased risk of GD (14, 15). mitogens and cause nonspecific activation of lymphocytes,
Similarly, other associations with genetic polymorphism in including self-reactive lymphocytes. Once self-reactive lym-
CTLA4 have been reported (16 18). How could this molecule phocytes are activated, they can continue to persist and ex-
be linked with increased susceptibility to GD? T Lympho- pand through antigenic stimulation from self-antigens. This
cytes constitutively express a costimulatory molecule called is because the strength of antigen presentation required to
CD28 on their surface. During the initiation of an immune sustain an anti-self response is lower than that required to
response, the T cells receive their first signal through their initiate an anti-self immune response. A number of bacteria-
TCR interaction with the MHC-peptide complexes on the derived proteins have now been identified as superantigens
antigen-presenting cells (APCs). However, naive lympho- and linked to Kawasaki disease (21), rheumatoid arthritis
cytes require a second signal to undergo activation that is (22), and other autoimmune diseases (23). Although the exact
delivered through CD28 upon its interaction with specific mechanism of their action is not known, superantigens have
ligands, B7.1 and B7.2 (i.e., CD80 and CD86, respectively), been postulated to facilitate development of autoimmune
expressed on the surface of APCs. Subsequent to activation, diseases by: 1) activation of a diverse population of T cells
T cells up-regulate CTLA4 expression on their surface. The with different fine specificities, including self-reactive T cells;
CTLA4, which is structurally related to CD28, not only in- and/or 2) polyclonal activation of B cells, including those
teracts with CD80 and CD86, but does so with a 100-fold that produce autoantibodies. Polyclonal activation of B cells
higher affinity. Because of higher affinity, CTLA4 competes resulting in production of antibody has been shown for My-
with CD28 for binding to CD80 and CD86 and causes down- coplasma arthriditis mitogen (24) and toxic shock syndrome
modulation of T cell responses due to negative signaling toxin-1 (25). Production of autoantibodies is thought to be
and/or lack of continued positive signaling because of com- mediated by bridging of superantigen to CD4 T cells and B
petition with CD28 for ligand binding. A defective interac- cells. Superantigens have been identified from culture su-
tion of CTLA4 with its ligands could result in a generalized pernatants from Yersinia enterocolitica (26) and Y. pseudotu-
defect in the down-modulation of immune responses. There- berculosis (27), and these infections have been linked to Re-
fore, although the observations on CTLA4 polymorphism are iters syndrome, reactive arthritis, GD, and ankylosing
of considerable interest, at this time it is not clear how a defect spondylitis (28). Although we have a much better under-
in this gene could specifically enhance immune response to standing of superantigens and the mechanisms by which
TSHR and susceptibility to GD. they activate lymphocytes, we know very little about the role
of Yersinia or Yersinia-derived superantigens and/or mito-
B. Environmental factors gens in different disease states including GD. It is possible
that Yersinia-derived T cell superantigens and B cell mitogens
Factors such as genetic background, age, and gender par- might act independently or in combination to activate T cells
tially account for the development of GD. As with other and/or B cells, resulting in preferential expansion of B cells
autoimmune diseases, an environmental factor has long been recognizing cross-reactive epitopes on TSHR and Yersinia.
suspected in the etiology of GD. If environmental factors Such B cells will not only produce autoantibodies reactive
such as bacteria are involved, how would such an agent with TSHR, but could also serve as efficient APCs that could
initiate an autoimmune response to a specific self-antigen? present TSHR peptides to self-reactive T cells and perpetuate
Cells other than APCs do not express MHC class-II and thus anti-TSHR responses.
are not capable of presenting antigens to CD4 T cells. Sim- Molecular mimicry is one of the most commonly invoked
ilarly, most cells show either little or no expression of co- and studied mechanisms for the induction of autoimmunity.
stimulatory molecules and thus fail to provide the second A finite probability exists for two proteins sharing common
epitopes formed either by a primary sequence of identical indicating that they may share stretches of common se-
amino acids or by noncontiguous amino acids adjacent by quences. However, to date, a causal relationship between
virtue of three-dimensional conformation, and resulting in Yersinia infection and the development of GD has not been
molecular mimicry. After infection, the host mounts a brisk fully established (41, 42).
response against the microbe, and this response might be-
come directed against a self-antigen that can be sustained
D. Cross-reactivity of Y. enterocolitica proteins with TSHR
even after the clearance of the microbial agent due to con-
tinued antigenic stimulation by the endogenous host protein. Several years ago, Seetharamaiah et al. (55, 56) expressed
This response then can expand due to epitope spreading and the extracellular domain of human TSHR (ETSHR) using a
eventually cause the disease. Molecular mimicry has been baculovirus expression system. ETSHR protein was purified
insinuated in other examples of autoimmunity including to homogeneity using HPLC, and upon refolding it could
HLA-B27 and Klebsiella in ankylosing spondylitis (29), my- bind TSH (55) and anti-TSHR antibodies in patient sera (56).
elin basic protein, and mouse hepatitis virus in experimental Mice immunized with ETSHR developed some clinical fea-
allergic encephalomyelitis (30), cardiac myosin, and cox- tures characteristic of GD, including high levels of antibodies
sackie virus B4 in myocarditis (31, 32) and others (33). Pri- to the TSHR, with a concomitant elevation in T4 levels (57).
mary amino acid sequence homology between self-peptides Using this recombinant ETSHR, cross-reactivity of Y. en-
and the foreign antigens as well as conformational similar- terocolitica with ETSHR was explored. Initial studies showed
ities have been demonstrated (30, 32, 33). that antibodies produced in rabbits and mice to purified
ETSHR reacted with envelope preparations from Y. entero-
C. Y. enterocolitica and GD colitica. This cross-reactivity was specifically blocked by pu-
rified ETSHR or an envelope preparation of Y. enterocolitica.
Due to seasonal trends and geographic variations in the Moreover, antibodies reactive with ETSHR were readily in-
incidence of GD (34, 35), it has been suggested that infectious duced in mice by immunizing with Y. enterocolitica, but not
agents might be involved in triggering the breakdown of with other Gram-negative or Gram-positive bacteria. These
tolerance to TSHR in patients with GD. In addition to the studies provided the first direct evidence that immunization
reports of increased susceptibility to infections in individuals with Y. enterocolitica can lead to the production of antibodies
who fail to secrete certain ABO blood group antigens into the capable of reacting with TSHR (39).
saliva (36, 37), serological evidence for a recent bacterial or Subsequently, the envelope protein(s) responsible for
viral infection has been reported in a high number of newly cross-reactivity to ETSHR was identified using anti-Yersinia
diagnosed patients with GD (38). antibodies that specifically bound to the ETSHR protein (50-
Y. enterocolitica has been postulated to play a role in the kDa band) on a Western blot. The two low-molecular weight
induction of GD via molecular mimicry (39 41). This spec- Yersinia envelope proteins (5.5 kDa and 8 kDa) that cross-
ulation is supported by studies reporting the presence of reacted with the ETSHR, TSHR-cross-reactive protein
antibodies against Y. enterocolitica in a high proportion of (TSHR-CRP), were gel purified (40) and used for immunizing
patients (i.e., 72 81%) with autoimmune thyroid disease (42 CBA/J, C57BL/6 and BALB/c mice. Sera obtained from
45). In addition, Y. enterocolitica has been shown to have these mice recognized both ETSHR protein and TSHR-CRP
binding sites for TSH (46), and antibodies isolated from GD in an ELISA and Western blot (58). Subsequently, it was
patients could inhibit TSH binding to Yersinia (47) and react shown that TSHR-CRP was mitogenic for spleen cells (58)
with a 64-kDa protein, which was thought to be the bacterial from C3H/HeJ mice, which are lipopolysaccharide nonre-
protein to which TSH binds (47). Moreover, antibodies raised sponders, and was pronase-sensitive (58). Cell proliferation
against thyroid membranes have been shown to bind Y. studies using B cell- and T cell-enriched populations showed
enterocolitica (48), and outer membrane proteins (YOPs) en- that these protein(s) are mitogenic to B cells, and not to T
coded by a virulence plasmid of Yersinia have also been cells, and could induce production of high levels of IL-6 and
linked to the induction of GD (49). Wenzel et al. (50) reported significant amounts of IgG and IgM by B cells. These results
that 72% of patients with GD and 81% of patients with re- indicate that TSHR-CRPs, which contain epitopes that cross-
current GD had antibodies to YOPs, whereas only 35% of react with TSHR, represent highly effective B cell mitogens,
unmatched controls had such antibodies, and YOPs could and the mitogenic activity was not due to lipopolysaccharide
induce antibodies in rabbits that recognized components of contamination. Alignment of peptide sequences showed that
thyroid epithelial cells. However, data reported by Arscott et TSHR-CRP is homologous to bacterial lipoproteins (LP). Al-
al. (51) suggested that there was no unique serological re- though, all Gram-negative bacteria produce LP, cross-reac-
activity against YOPs or other Yersinia membrane antigens tivity of Y. enterocolitica LP with TSHR was unique (59).
and that any causal relationship between Yersinia infection Moreover, LP was capable of inducing high levels of IL-6 and
and GD may be related to activation of cross-reactive T cells IL-8 production by human B cells. These observations have
leading to breakdown of self-tolerance to TSHR. Further- significant implications for understanding the role of mo-
more, Ebner et al. (52) reported that rats immunized with Y. lecular mimicry in the breakdown of self-tolerance and gen-
enterocolitica-purified YOPs developed thyroiditis. In this re- erating autoimmunity to TSHR (59).
gard, T cell-mediated immunity toward Y. enterocolitica has In a recent review, Benoist and Mathis (60) raise some
also been reported in patients with GD (53). Burman et al. (54) important issues to consider with regard to the role of mi-
reported that TSHR-specific cDNA probe could hybridize to crobial agents in promoting T cell-mediated autoimmunity.
DNA from Y. enterocolitica under low stringency conditions, Although a number of animal models have provided con-
vincing evidence that a given autoimmune disease can be for the regulation of anti-TSHR antibody production (79, 80),
induced by a particular microbe, they argue that many of the other studies have reported contradicting results (81 83).
associations are less convincing based upon five different Because of the unavailability of purified TSHR, the antigen
criteria they propose for air-tight cases (which are modi- specificity of T cells could not be determined, and thus it is
fications of previously presented concepts). Although they difficult to assess the significance of changes in the total T cell
raise some important points with regard to the role of mo- ratios. More recent studies have analyzed the TCR gene
lecular mimicry for us to consider, their perspective is re- utilization in lymphocytes infiltrating the thyroid gland (84,
stricted to issues involving T cell epitope recognition and 85). The rationale for these studies was that cells infiltrating
diseases in which T cells, and not B cells, act as effector cells. the thyroid gland are likely to be thyroid antigen-specific and
Moreover, it should be noted that molecular mimicry is only that utilization of a restricted number of TCR genes by these
one of several potential mechanisms that a microbe can use infiltrating T cells would suggest that a limited number of T
to induce autoimmunity. As discussed in Section II.B, micro- cell epitopes are involved. Data from these studies showed
bial agents can cause immunological perturbations through that early after onset of GD there is preferential usage of
their mitogenic activity, ability to alter self-proteins and/or certain TCR variable (V) genes (84). However, this study
induce proinflammatory cytokines, and direct up-regulation and others also showed that there was considerable variation
of MHC gene products and costimulatory molecules (by- in TCR usage among different patients (84, 86). This is not
stander effects). Needless to say, as in all other diseases, surprising, considering the fact that T cells recognize anti-
including metabolic disorders, cancer, etc., it is not easy to gens in the context of MHC molecules. Because different
directly demonstrate the role of environmental factors in patients might have different HLAs, they need different
autoimmunity. As argued by Benoist and Mathis (60), more TCRs to recognize the antigenic peptide (87). The HLA vari-
experimental evidence is needed to fully establish the role of ation, coupled with the lack of information on antigen spec-
a given microbial agent in the initiation of autoimmunity and ificity of infiltrating cells, makes the interpretation of these
autoimmune diseases. With the availability of an animal data difficult. A putative role for T cells in GO will be dis-
model, now it might be feasible to investigate the potential cussed later.
immunomodulatory effects of Y. enterocolitica and/or the Passive transfer of Igs from GD patients to experimental
consequence of molecular mimicry that it exhibits with TSHR animals causes increased thyroid hormone production (88).
on the development of GD. However, efforts to induce anti-TSHR antibody response
using thyroid membranes have largely failed (89). Because of
the low abundance of TSHR on thyroid cells (103 to 104
receptors per cell) (90) as well as instability of the TSHR
III. Immunological Basis for GD
protein (91), it has been very difficult to purify the TSHR from
GD is one of the most common endocrine disorders. Al- thyroid membranes. Thus, to date, it has not been feasible to
though the etiology of GD is not known, it is widely accepted fully evaluate the importance of the idiotypic network for the
to be an autoimmune disease. Patients with GD have circu- regulation of TSHR-specific antibody production. Based on
lating autoantibodies directed against the TSHR, and there is studies published thus far, there exists little or no evidence
overwhelming evidence that hypersecretion of thyroid hor- to suggest that an idiotypic network plays a critical role in the
mones is mediated by binding of anti-TSHR autoantibodies regulation of immune responses to TSHR.
to the TSHR on thyroid membranes (61 67). In addition to Recently, considerable efforts have been made to under-
stimulatory TSHR-antibodies (TSHR-Abs), antibodies that stand the role of apoptosis in both Hashimotos thyroiditis
inhibit either basal adenylate cyclase activity (68 70) or TSH- and GD. It appears that thyroid-infiltrating lymphocytes in
mediated production of cAMP (blocking TSHR-Abs) have patients with Hashimitos thyroiditis express little or no Fas
also been described in patients with GD (71). Antibodies with but exhibit high levels of Bcl-2, whereas their thyrocytes
these different biological properties can be detected in an in show decreased Bcl-2 and increased Fas as well as Fas ligand.
vitro assay using cells expressing TSHR. Increased cAMP This would render thyrocytes susceptible to Fas-mediated
production by TSHR-expressing cells when cultured with Igs apoptosis and facilitate thyroid destruction. However, in
from patients with GD would indicate the presence of stim- contrast, patients with GD show increased Bcl-2 and de-
ulatory antibodies, whereas inhibition of TSH-mediated increased Fas expression on their thyrocytes, while exhibiting
crease in cAMP production would indicate the presence of increased Fas and decreased Bcl-2 on their lymphocytes. This
blocking TSHR-Abs (72). Earlier studies have shown that the would lead to apoptotic death of infiltrating lymphocytes
stimulatory TSHR-Abs are restricted to the IgG1 subclass (73, and sparing of thyroid from the effects of inflammatory
74), but blocking TSHR-Abs or antibodies directed against responses. Recent papers (9294) on this topic have discussed
other thyroid antigens (e.g., thyroid peroxidase) are not re- the role of apoptosis in thyroid autoimmunity; therefore, we
stricted and might arise secondary to thyroid tissue damage will refrain from a detailed discussion here.
(75, 76). Some patients may have both stimulatory and block-
ing TSHR-Abs, and the net biological effect of the serum
might be influenced by the relative concentrations of these IV. Autoimmune Responses to Thyroid Autoantigens
antibodies (73, 77). Although, in general, patients with more
severe disease have the highest levels of stimulatory TSHR- Antibodies to TSHR either exhibit no functional effect on
Abs, it is not necessarily true in all cases (78). the thyroid or can stimulate or block TSH-mediated activa-
Although, imbalances in T cell ratios have been proposed tion of the thyroid. Depending upon the relative levels of
blocking and stimulatory antibodies, the disease can mani- portant conclusions. Both TSH and autoantibodies can pri-
fest itself as either hypothyroidism or hyperthyroidism. Al- marily bind to the ETSHR, and the TSH binding epitope
though most cases of hypothyroidism are due to thyroid consists of discontinuous amino acids that come together due
destruction, either due to apoptosis or direct T cell-mediated to protein folding. Antibodies against one or more epitopes
damage, seen in Hashimotos disease, some cases of hypo- can inhibit TSH binding or TSH-mediated activation of cells
thyroidism are due to high levels of thyroid stimulation by affecting a step subsequent to TSH binding. The stimu-
blocking antibodies (TSBAbs). TSBAbs can either block TSH latory autoantibody binding sites reside predominantly at
binding or affect a step subsequent to TSH binding and the N terminus of the protein, whereas the blocking anti-
prevent thyroid activation. In contrast, most cases of GD are bodies bind primarily to the C terminus. Glycosylation of the
characterized by the presence of stimulatory antibodies. This receptor is important for proper folding of the receptor and
observation was based upon early studies that demonstrated autoantibody binding. However, it is not clear whether au-
the presence of long-acting thyroid stimulators in the sera of toantibodies directly interact with the sugar moieties or with
patients with GD, which were subsequently shown to be epitopes that are formed as a result of proper glycosylation
present in the Ig fractions. These studies clearly established of the receptor protein. A number of excellent reviews (99
that GD is an autoimmune disease and that antibodies 103, 106) have dealt with the structure-function relationship
against TSHR are the cause. Based on these very early ob- of TSHR; therefore, we will not discuss this issue any further
servations, various assays to detect autoantibodies to TSHR in this paper.
were developed and have been used for the diagnosis of
hyperthyroidism associated with GD.
In 1989, a cDNA-encoding dog TSHR was cloned (95)
V. TSH Receptor as an Autoantigen
followed by cloning of human and rat cDNAs (96 98). This
resulted in numerous studies aimed at understanding the The TSHR belongs to a family of G protein-coupled re-
structure-function relationship of the protein aimed at the ceptors and is structurally very similar to other polypeptide
determination of various binding sites for TSH as well as hormone receptors including for FSH and chorionic gonad-
functionally different antibodies (99 103). Many initial stud- otropin/LH. Despite their structural similarity, it appears
ies used synthetic peptides that were derived from the pro- that only TSHR can serve as an effective autoantigen. This
tein sequence predicted from the cDNA sequence and pro- has raised the question as to whether unique features of the
vided some insights into the potential antibody binding TSHR might render the protein a ready target for autoim-
epitopes (99 103). Initial efforts to express the protein in mune attack. TSHR undergoes posttranslational cleavage
bacteria resulted in limited yields of protein that was not and forms a two-subunit structure (107, 108), and this could
folded properly. Subsequent studies using an insect cell ex- result in the generation of self-antigens (109). If the A subunit
pression system resulted in higher yields of both glycosy- is cleaved and is picked up by APCs, it is conceivable that
lated and nonglycosylated proteins representing the ETSHR. TSHR peptides can then be presented in the context of MHC
Studies utilizing these protein preparations clearly showed class-II molecules. This could lead to the activation of anti-
that the ETSHR was sufficient for the TSH and patient au- gen-specific CD4 T cells that have escaped negative selec-
toantibody binding, and that glycosylation was required for tion in the thymus and are in the periphery. Such activation
autoantibody recognition (104, 105). Although a small frac- could result in breakdown of self-tolerance to TSHR and
tion of the protein produced in insect cells acquired appro- result in the eventual production of stimulatory antibodies.
priate conformation, as indicated by TSH and autoantibody In addition to the subunit structure of TSHR, other im-
binding, this expression system was unsuitable for the pro- portant immunological factors must be required to induce an
duction of TSHR with native conformation because the pro- autoimmune response. Although self-reactive T cells might
tein remained largely aggregated. Therefore, many studies, escape negative selection in the thymus, they can be kept in
aimed at determining the functionally relevant epitopes on an inactive state through a number of different peripheral
TSHR were conducted using two major approaches involv- tolerance mechanisms (12). For an effective antigen presen-
ing transfection of mammalian cells with various cDNA con- tation, not only does the antigenic peptide have to be pre-
structs. One involved construction of TSHR/LH/choriogo- sented by the MHC molecule, but the strength of signal is also
nadotropin receptor chimeras in which select segments of very important. This is referred to as the first checkpoint, is
TSHR were replaced with the corresponding segments from demonstrated in animal models of diabetes, and is a conse-
the LH/choriogonadotropin receptor. Studies from this ap- quence of suboptimal presentation of antigens (110, 111).
proach resulted in the identification of regions of TSHR that Even if an appropriate signal is delivered, it has to be ac-
are critical for TSH and blocking and stimulatory antibody companied by a second signal to activate naive T cells. It is
binding. Another approach has been to use deletion mutants generally accepted that self-peptides do not efficiently acti-
and/or site-directed mutagenesis to identify the functionally vate APCs (i.e., lack of costimulation) and thus can induce T
relevant regions of TSHR. These studies revealed that certain cell anergy rather than activation (111, 112). An even more
glycosylation sites and cysteine residues are absolutely es- important mechanism is the engagement of CTLA4, which
sential for proper folding of the receptor. Because of limita- down-modulates the immune response (113118). More re-
tions associated with each of the approaches mentioned cently, another molecule called PD1 (programmed cell death)
above, no single approach has yielded conclusive results on has been shown to suppress cytokine production through
the overall structure-function relationship of the TSHR. cell cycle arrest and induce T cell anergy (119 124). Yet
However, collectively, these studies have led to several im- another mechanism that can help maintain tolerance in the
face of appropriate antigen presentation is phenotypic skew- tive TSHR (55). These studies suggested that the ectodomain
ing of helper T cells (125128). There are two types of helper was sufficient for TSH binding, albeit at a lower affinity, and
T cells, namely Th1 and Th2. Cytokines produced by Th2 for the induction of TSHR-specific antibodies. To identify a
cells play a more important role in antibody production than strain of mice susceptible to the development of hyperthy-
the cytokines produced by Th1 cells. Therefore, if Th1 cells roxinemia, C57Bl/6J, BALB/CJ, B10BR/SgSnJ, and SJL/J
instead of Th2 cells are activated, it is unlikely that it will mice were immunized with ETSHR (57). Sera obtained after
result in a pathogenic antibody response against the TSHR. five immunizations from different strains of mice were TBII-
If appropriate self-reactive T cells are activated, still they can positive, albeit at different levels of significance (57), whereas
be deleted by activation-induced cell death (129 133). Self- only sera collected on d 55 from BALB/CJ mice showed
antigens are not readily cleared, and therefore they can pro- significant elevation in T4 hormone compared with sera ob-
vide repetitive and continuous stimulation to T cells, causing tained from the corresponding control mice. However, the T4
them to undergo activation-induced cell death through Fas elevation was transient and indicated that immunization
ligation by Fas ligand. Therefore, yet unknown perturbations with ETSHR had failed to break tolerance to self-TSHR and
in the immunoregulatory mechanisms of the host might con- that immunization using TSHR with native conformation
tribute to the pathogenesis of GD. may be required.
To address this, BALB/CJ mice were primed with the
ETSHR and then challenged with solubilized thyroid mem-
VI. Evolution of Autoimmune Response to TSHR brane. Relative to controls, immunized mice showed high
Unlike immune responses against exogenous antigens, levels of TBII activity and significantly elevated T4 (57). The
which can be readily monitored, evolution of immune re- [131I] uptake by thyroid glands of test mice was found to be
sponses, from the initiation to the clinical onset of the disease, higher than in control mice, suggesting that the elevations in
against self-antigens is impossible to monitor in humans. free T4 were not a consequence of gland destruction. His-
Therefore, our current understanding of the pathogenesis of topathological examination of thyroid glands from hyper-
most human autoimmune diseases is based on studies from thyroid mice showed morphological alterations characteris-
experimental animal models. There is no appropriate animal tic of hyperactive glands, including hydropic and subnuclear
model in which autoimmune GD develops spontaneously. In vacuolar changes, as well as focal scalloping of the colloid
the past, efforts to develop an animal model have not been within isolated acini. No inflammatory activity, interstitial
successful because of the difficulty in purifying TSHR from fibrosis, acinar atrophy, or glandular destruction was found,
thyroid membranes in sufficient quantities required for the and this further suggested that the elevated T4 levels were
induction and characterization of experimental autoimmune due to hypersecretion. These results showed that ETSHR can
GD. Since cloning of the receptor, many investigators have prime the animal and allow development of antibodies that
tried to establish an animal model for GD, and they have met can cause hyperthyroidism. The limitation of this study was
with varying success. The earliest studies used synthetic that the mice became euthyroid within weeks after cessation
peptides and various bacterial and insect cell-derived proof TSHR injection, again indicating failure to break tolerance
teins to immunize mice. In all cases, the animals responded to self-TSHR.
with antibody production against the immunizing antigen In an attempt to develop an animal model for GD, Marion
and in some cases against the native TSHR as detected by the et al. (134) used affinity-purified human TSHR from a human
appearance of the TSH-binding inhibitor Igs (TBII) and/or thyroid cell clone designated GEJ to immunize B10S (H-2s),
blocking antibody activity. Some of these studies reported B10 (H-2b), B10G (H-2q), B10 D2 (H-2d), and B10BR (H-2k)
production of stimulatory antibodies; however, those reports male and female mice. The male and female H-2s, male H-2b,
could not be independently confirmed by others. and female H-2q mice exhibited lymphocytic infiltration in
TSHR is a member of the large family of guanine-nucle- their thyroid glands. Immunized mice showed transient mar-
otide binding (G) protein-coupled receptors. The full-length ginal elevation in T3. Because no data on serum TBII, TSBAb,
TSHR cDNA consists of an open reading frame encoding 764 and thyroid-stimulating antibody (TSAb) activities were pro-
amino acids, including a 21-amino acid signal peptide, a vided, it was not possible to conclude whether these mice
large extracellular domain, seven transmembrane domains, developed hypothyroidism or hyperthyroidism.
and a short cytoplasmic tail (9598). The ETSHR forms the Other investigators have attempted to develop an animal
binding site for TSH on the outside surface of the thyroid cell model for autoimmune GD (hyperthyroidism) and thyroid-
membrane, and the cytoplasmic domain interacts with the itis (hypothyroidism) using recombinant TSHR. Costagliola
regulatory subunits of adenylate cyclase. Initially, high levels et al. (135) used a fusion protein consisting of ETSHR and
of the nonglycosylated ETSHR were produced using a bac- maltose binding protein (MBP-ECD). Sera or IgG from im-
ulovirus expression system (55). This protein was purified munized mice did not show any thyroid-stimulating activity
to homogeneity using reversed phase HPLC. The purified (TSAb), but showed significant TBII and TSBAb activity and
ETSHR protein was refolded and shown to specifically bind lower serum T4 levels compared with mice immunized with
125
I TSH in a dose-dependent manner (56). MBP alone. Histological examination of thyroids revealed
extensive vascularization and an atypical lymphoblastoid
A. Early studies on pathogenesis of experimental GD infiltration. In a subsequent study, they extended these ob-
servations to male and female mice (136). In another study,
The insect cell-derived protein was used to successfully they immunized female NOD (H2g), CBA (H2k), and C57BL
produce antibodies capable of blocking TSH binding to na- (H2b) mice with MBP-ECD (137). No significant serum TBII,
TSAb, and TSBAb were detected in any of the strains of mice. antigen (MBP) to elicit a response against the self-antigen? It
Although there was no lymphocytic infiltration of the thy- is well known that a foreign antigen is likely to be more
roid glands of CBA or C57BL mice, all NOD mice had severe immunogenic than a self-antigen such as TSHR. If this were
thyroiditis. Based on these studies, the authors concluded the case, it is likely that the immune response will be largely
that H2d mice develop thyroiditis and TBII/TSBAb, H2g directed against the MBP. In the absence of a clear demon-
mice develop thyroiditis in the absence of functional TSHR stration of TSHR specificity of the T cells in MBP-TSHR
antibodies, whereas H2b and H2k mice are resistant. Fur- immunized mice, the possibility of MBP-specific T cells pro-
thermore, they showed the ability of TSHR-primed spleno- viding help to TSHR-specific B cells, through linked recog-
cytes to transfer thyroiditis to naive recipients (138). How- nition, cannot be ruled out.
ever, neither the antibody properties nor the hormonal Carayanniotis et al. (143) attempted to develop an animal
perturbations were consistent with findings in GD. In many model by immunizing C57Bl/10, B10.BR/SgSn, C3H/He,
of these studies, although immunization with MBP alone had SJL, DBA/I, and BALB/c mice with an ETSHR protein pro-
no significant effect, the influence of the MBP component of duced in insect cells. All strains of mice showed strong spe-
MBP-ECD on the induction of thyroiditis was not evaluated. cific T cell proliferative responses. Sera from SJL and BALB/c
One possible explanation is that because more recent studies mice showed good TBII activity. However, there were no
have shown that glycosylated ETSHR is required for stim- significant changes in serum TSH, T4, and iodine uptake.
ulatory antibody recognition (and presumably for its induc- Authors suggested that their inability to induce hyperthy-
tion), antibodies raised against the nonglycosylated ECD roidism is most likely due to the inappropriate folding of the
produced in bacteria might not have been sufficient to ETSHR protein. Vlase et al. (144) immunized female BALB/c
cause GD. and CBA mice with human TSHR-ECD produced in Esche-
Both BALB/c and NOD mice were immunized with MBP- richia coli and in insect cells, respectively. Similar to Caray-
ECD, and unfractionated T cells or CD4-enriched cells ob- anniotis et al. (143), these investigators failed to induce either
tained from these mice were stimulated in vitro with the hyperthyroidism or thyroiditis. Subsequently, studies from
MBP-ECD protein and then adoptively transferred to the a number of laboratories have shown that insect cells are not
corresponding strain of naive recipients. The recipients capable of removing the human signal sequence from the
showed TBII activity in their sera that persisted for 12 wk. protein. Because the cDNA used for protein expression in
NOD mice showed marginal hypothyroidism, whereas most these two studies encodes for a human signal peptide con-
BALB/c mice were euthyroid with few showing some ele- sisting of 21 largely hydrophobic amino acids at the N ter-
vation in T4 levels (138). Approximately two thirds of minus, it likely affected the conformation of the protein that
BALB/c mice showed accumulation of adipose tissue and may be needed for pathogenic antibody production. In an-
infiltration of mast cells in the orbital tissue. Collectively, other study, Vlase et al. (145) used refolded ectodomain of
these studies showed that multiple factors determine the mouse TSHR produced in insect cells to immunize BALB/c
outcome of immunization with TSHR, and suggested the mice. Immunized mice developed considerable TBII and
polygenic nature of the disease (139). TSBAb activities and exhibited lower levels of T3, but not T4,
It is apparent that an effective antibody response (i.e., IgG) and higher levels of TSH.
against the TSHR protein would require help from T cells
that have the same antigen specificity (140 142). This phe- B. Recent studies on the pathogenesis of experimental GD
nomenon is called linked recognition and would require
antigen presentation by professional APCs and activation of Shimojo et al. (146) immunized female AKR/N (H-2K)
antigen-specific T cells before autoantibody production by B mice with fibroblasts expressing both MHC class-II and a
cells with specificity for the same antigen. However, it is full-length human TSHR. Most of these mice exhibited good
interesting to note that the epitopes recognized by T cells and serum TBII activity. About 20% of these mice developed
B cells need not be part of the same protein but have to be hyperthyroidism as evidenced by significantly elevated se-
physically linked (140 142). This is important for subsequent rum T4 levels. This was similar to earlier findings of severe
antigen presentation by B cells because these cells will in- hyperthyroidism when BALB/c mice were inoculated with
ternalize the antigen through receptor-mediated endocyto- ETSHR followed by thyroid membrane preparations (57).
sis, process the antigen, and present specific peptides on their Based on these and other results (146 148), the authors have
surface leading to subsequent antigen-specific T cell activa- suggested that expression of MHC class-II on cells that ex-
tion. This principle has been exploited in the development of press TSHR can result in the induction of stimulatory anti-
an effective vaccine against Haemophilus influenzae type B. TSHR antibodies in a small proportion of animals. The data
The protective antibodies against this bacterial infection are presented in this study and an earlier study (57) may also
directed against the bacterial polysaccharides. Adults readily suggest that some of the pathogenic epitopes might be
produce such protective antibodies, whereas infants are not present on the native TSHR. A similar study from another
as efficient. Therefore, the bacterial polysaccharide is linked group (149) confirmed these observations by showing that
to the tetanus toxoid against which the infants mount an approximately 25% of the immunized animals developed
effective response. In this case, B cells that bind to the poly- hyperthyroidism and one mouse developed hypothyroidism
saccharides can readily produce antibodies with help from T with elevated TSH levels. Mice with hyperthyroidism
cells specific for peptides from tetanus toxoid because of the showed thyroid hypertrophy without lymphocytic infiltra-
linked recognition. In light of this, what is the consequence tion. When mice were immunized with cells expressing
of using a self-antigen (TSHR) that is fused to a foreign TSHR and MHC class-II along with alum as an adjuvant, they
showed increased prevalence of the disease (approximately observation (153). This seems reasonable in a T cell-mediated
45%), with a shorter duration required for the onset. How- autoimmune disease that results in the destruction of the
ever, when TSHR along with complete Freunds adjuvant target tissue because the effector T cells need to recognize the
was used, approximately 30% of the animals developed the self-peptide/MHC complex on the target tissue. It is unlikely
disease with a slower onset. In this study, no apparent dif- that such an aberrant expression of class-II is required for the
ference was found between male and female mice (149). induction of an autoantibody response. This proposition is
Additional studies showed that several different strains of based on the current understanding of T cell and B cell
mice with different non-MHC background but expressing interactions required for an effective antibody production
H-2K haplotype were able to produce TBII antibodies upon (Fig. 1). The initial step in an immune response against a T
immunization with cells expressing class-II and TSHR pro- cell-dependent antigen is phagocytosis of the antigen in the
teins (148). Unlike the earlier report with AKR mice (147), periphery by professional APCs, most likely dendritic cells
these studies showed that TBII activity could be induced in (DCs). Once they pick up the antigen, DCs migrate and begin
CBA and C3H mice using fibroblasts expressing TSHR with- processing the antigen in the T cell zone of the regional
out the class-II protein. A subsequent study showed that the lymph node. Here, antigen-specific T cells, which enter the
N-terminal half of the ectodomain might be sufficient to lymph node through the afferent lymphatics, are trapped by
induce TBII activity in AKR mice, but the entire ectodomain the DCs with relevant peptide-MHC class-II on their surface.
was needed to induce TSAb activity in approximately 20% of Subsequently, as they circulate through the lymph nodes,
the animals. To carry out systematic studies on the molecular antigen-specific B cells are trapped where they are activated
mechanisms, a higher proportion of animals need to develop by antigen-specific T cells to produce antibodies. Antibodies
the disease, and that may require a more susceptible strain enter the circulation and react with the TSHR, causing hy-
of mice, different antigen preparation, and/or immunization perthyroidism. In this scenario, it is likely that the availability
schedule (150, 151). of appropriately folded protein to B cells might be more
Results from studies using fibroblasts stably transfected important than the expression of class-II molecules on the
with human TSHR and MHC class-II have been used to argue cells used for immunization. Moreover, a large number of
that aberrant class-II expression is required for disease in- studies have clearly shown that there is little or no priming
duction (146). Although this might be correct, there are no of naive T cells in the absence of costimulation. Because these
conclusive data to support this conclusion. The contention RT cells most likely lacked both adhesion and costimulatory
that aberrant expression of class-II is necessary has been molecules (i.e., B7.2), it is not apparent how these cells could
based largely on finding that MHC class-II is expressed in have served as efficient APCs to prime naive T cells required
both thyroid and islets of affected patients (152). Subsequent for optimal anti-TSHR responses.
studies showed that expression of class-II molecules or ex- Some studies have shown that certain fibroblasts, includ-
ogenous antigens on -islet cells using a rat insulin promoter ing RT cells, can express B7.1. Cells expressing B7.1 mole-
can result in varying outcomes, including development of cules are most likely to elicit Th1 rather than Th2 type of
spontaneous diabetes, and thus lent support for the initial response. This assertion is based on recent findings which
FIG. 1. Antigen-processing pathway

that might be required for autoantibody
production against the TSHR. Note the
consequence of antigen processing by
both the endogenous and exogenous
pathways. Even when the antigen is ex-
ogenously processed, activation of Th2
cells might be required for an optimal
antibody production.
show that B7.1 and B7.2 preferentially activate Th1 and Th2 showed hypertrophy with no cellular infiltration, and the
type of responses, respectively (154). Because it is generally extraocular muscles were normal.
believed that Th2 type of cytokines play an important role in Another study reported successful induction of Graves-
GD, it is unlikely that B7.1 expression on RT cells could like disease in nearly 100% of female BALB/c mice using
facilitate such a response. MHC class-II molecule is a het- either mouse or human TSHR proteins (160). BALB/c mice,
erodimer made up of an -chain and a -chain. Each of the known to generate Th2-type responses, were used because
chains consists of two domains. RT cells constitutively do not antibody-mediated autoimmune diseases are currently
express class-II molecules and are transfected with a cDNA thought to be driven by Th2 helper T cells. Female BALB/c
that can encode a chimeric class-II molecule. The -1 and -2 mice were used because of their expected enhanced suscep-
and -2 domains of this chimeric class-II were derived from tibility to GD. The protein was either expressed on the sur-
H-2K haplotype, and the -1 domain was derived from H-2D face of syngeneic or xenogeneic cells or produced in a soluble
(155). These cells were originally constructed to identify pep- form. Irrespective of the antigen used for immunization,
tide binding sites on class-II and were used in vitro as APCs nearly 100% of the mice developed hyperthyroidism char-
to test for proliferative responses of T cells primed in vivo by acterized by the presence of high levels of TBII, TSAb, T4, and
direct antigen inoculation. However, it is not clear whether T3. This study was the first to use mouse TSHR to induce the
the chimeric class-II molecule expressed on RT cells could disease in mice. Mice were equally susceptible to immuni-
have served as an alloantigen (due to -1 domain derived zation with either a full-length human TSHR expressed on
from H-2d) in AKR mice (H-2 k haplotype) leading to acti- 293 human cells (xenogeneic cells that are histoincompatible)
vation of alloreactive T cells rather than TSHR-specific T or purified ectodomain of the human TSHR. This showed
cells, that can provide nonspecific help. This might explain that the cells expressing the TSHR need not act as APCs and
a lack of T cell infiltration in thyroids of mice with severe that all necessary T and B cell epitopes are present on the
hyperthyroidism. Nevertheless, these studies are very im- ectodomain. Similarly, mice immunized with m12 cells (a B
portant because they clearly showed that hyperthyroidism lymphoblastoid cell line with H-2D MHC haplotype, same as
can be induced in experimental animals using a recombinant that of BALB/c mice) and expressing either mouse or human
TSHR. Moreover, they showed that the nature of immune TSHR developed the disease with a similar time of onset,
frequency, and severity. In addition, some groups of mice
response is directly dependent on the antigen and/or the
showed a reduction in body weight and thyroid infiltration
adjuvant used for inoculation. Thus, immunization using
of lymphocyte. In this model, development of the disease is
TSHR with native conformation might result in the produc-
a slow and progressive process with antibodies against
tion of stimulatory antibodies that could then cause
TSHR that can be detected only by ELISA, appearing at
hyperthyroidism.
approximately 4 6 wk after initiation of antigen adminis-
Other studies have reported using cDNA vaccination to
tration. This is followed by the appearance of TBII activity by
induce pathogenic antibodies against the TSHR. These stud-
d 60 80 and TSAb by d 150, with clinical onset of the disease
ies yielded somewhat contradictory results depending upon
by d 250 or later.
the strain of mice used for immunization. BALB/c mice Why do the pathogenic antibodies (i.e., TSAbs) develop
immunized with the cDNA developed antibodies to TSHR late in the immune response and result in a delay in the onset
(156). Only one mouse contained TSAb, but all mice, irre- of the disease? Although the answer remains uncertain, it is
spective of their autoantibody status, were euthyroid. Ex- most likely due to the time required for affinity maturation
amination of the thyroid glands from these mice revealed and/or repertoire spreading eventually leading to the pro-
nondestructive thyroiditis, with B cell infiltration. When a duction of pathogenic antibodies. Although the reasons for
similar experiment was carried out using NMR outbred the delay are not known, we speculate that TSAbs might have
mice, all mice developed antibodies to TSHR detected by to interact with TSHR with a relatively higher affinity,
fluorescence-activated cell sorter (157). Nine of 30 males and/or with specific epitopes to mediate their TSH agonist
showed mild hypothyroidism with TSBAb and lower T4, activity. It is well known that affinity maturation is largely
whereas four of 30 females showed signs of hyperthyroidism dependent upon somatic hypermutation of the Ig genes,
with elevated T3 and T4 levels and TSAb activity with a which in turn is dependent on DNA replication and cell
concomitant decline in TSH levels. Another study showed a division. This would imply slow and continuous evolution
heterogeneous response upon vaccination of wild-type and of anti-TSHR antibody responses toward the production of
interferon- (IFN-) / BALB/c mice with TSHR cDNA higher affinity antibodies. Alternatively, these results sug-
(158). Further refinement of this approach and the use of gest that antibody specificity might gradually spread from
female mice might lead to the development of an animal one that is restricted against immunodominant epitopes to
model in which a higher proportion of mice develop GD. include cryptic epitopes. This later phenomenon of epitope
In a more recent study (159), different strains of mice were spreading has been clearly shown in other autoimmune dis-
immunized with adenovirus expressing either TSHR or - eases (161164). In this scenario, the initial immune response
galactosidase three times at 3-wk intervals, and mice were against a self-antigen is mostly directed against the dominant
observed for 8 wk after the third immunization. Fifty-five epitopes (operationally defined) and is not pathogenic. How-
percent of female and 33% of male BALB/c mice and 25% of ever, as the immune response evolves, it may become di-
female C57BL/6 mice developed hyperthyroidism charac- rected against pathogenic cryptic epitopes. Should immu-
terized by the presence of TBII and TSAb activity and ele- nodominant epitopes become pathogenic, deleterious
vated T4 levels in the sera. Thyroid glands of affected mice consequencess would be anticipated. Therefore, for the sur-
vival of the species, it is imperative that the pathogenic ence of antibodies directed against thyroid antigens such as
epitopes remain cryptic. Efforts are under way to use a re- TSHR or thyroid peroxidase (168). Regardless of whether
ceptor preparation that has the stimulatory antibody binding hyperthyroidism occurs first, the signs and symptoms of GO
sites but is devoid of the immunodominant regions of the become manifest in 85% of patients within 18 months (169).
receptor, which are irrelevant for TSAb binding or induction, Symptoms described by patients with GO include a gritty
to determine whether the disease can be induced within a sensation in the eyes, sensitivity to light, increased tearing,
shorter duration. Our unpublished studies suggest reper- double vision, blurring of vision, and feeling a pressure sen-
toire spreading as indicated by the change in the TCR V and sation behind the eyes (165). On physical examination, ex-
the TSHR peptide specificity of TSHR reactive T cells during traocular muscle dysfunction, proptosis (forward protrusion
the evolution of the immune response. Further studies are of the eyes), periorbital and eyelid edema, conjunctival che-
required to determine whether both B cell repertoire spread- mosis (swelling) and injection (redness), lid lag and retrac-
ing and somatic hypermutation contribute to the evolving tion (or stare), or exposure keratitis (corneal injury due to
immune response against the TSHR. dryness) may be detected. Most patients experience only the
minor congestive signs of GO (chemosis, injection, lid
edema), with improvement in several months without treat-
VII. Graves Ophthalmopathy ment. In a minority of patients, however, the disease
A. Clinical features progresses, and one or more of the components (proptosis,
extraocular muscle dysfunction, and periorbital edema) may
In addition to hyperthyroidism, over 2550% of individ- be severe and symptomatic for several years. In rare in-
uals with GD have clinical involvement of the eyes known stances, patients develop compressive optic neuropathy with
as thyroid-associated ophthalmopathy (TAO) or GO (Fig. 2; decreased visual acuity, dulling of color perception, visual
Ref. 165). Although some patients experience only mild oc- field defects, and rarely, blindness.
ular discomfort, 35% of patients suffer from intense pain
and inflammation with double vision or even loss of vision. B. Pathological findings
In addition, a small percentage of patients with GD have
clinically apparent pretibial dermopathy, a diffuse or nod- Many of the clinical symptoms and signs of GO can be
ular thickening of the skin on the anterior lower leg (Fig. 3). explained on a mechanical basis by an increase in the volume
These skin changes occasionally occur on other parts of the of both the orbital fatty connective tissues and the extraocular
body, often after local trauma. Although eye and pretibial muscle bodies (170). This expanded tissue volume within the
skin changes are clinically obvious in the minority of patients orbit in GO can be measured in computed tomography scans
with GD, subtle eye and skin changes can be detected using taken of GO patients orbits (171). Some patients appear to
orbital or dermal ultrasonography, or other sensitive detec- have enlargement of extraocular muscles as the predominant
tion techniques, in the majority of patients (166, 167). Con- anatomical change. Others exhibit little eye muscle involve-
versely, although approximately 10% of patients with GO do ment but show significantly increased orbital adipose tissue
not have hyperthyroidism, the majority have laboratory ev- volume. Histological examination of extraocular muscles re-
idence of thyroid autoimmune disease, including the pres- veals intact muscle fibers that are widely separated by edem-
FIG. 2. Patient with severe GO. Proptosis,

lid retraction, conjunctival erythema, and
periorbital edema are evident.
FIG. 3. Histological examination of or-

bital tissues from a patient with GO. A
mononuclear-cell infiltration is seen
within the adipose and connective tis-
sue compartments. Magnification,
250.
atous connective tissues (172). These perimysial tissues (179), TCR -chain (TCR-; Ref. 180), and Ig heavy chain
contain excess glycosaminoglycans (GAGs) that appear com- have been shown in association studies to confer suscepti-
posed predominately of hyaluronan and chondroitin sulfate bility to GD, but at small relative risk. A few studies specif-
(173). A similar accumulation of GAGs, with more profound ically examined genetic differences between GD patients
adipose tissue expansion, is apparent in the fatty connective with GO and GD patients having no apparent eye disease.
tissue compartments of the posterior orbit (Fig. 4; Ref. 174). However, these did not yield any confirmed susceptibility
It remains likely that both the accumulation of GAGs and the loci for GO because the various polymorphisms offering
expansion of the adipose tissues contribute to the increase in susceptibility to GD were found in equal proportion in
orbital tissue volume characteristic of GO. However, further Graves patients with and without eye disease (181184).
quantitative studies are needed to reliably partition the net More recent whole genome linkage studies suggested that
effects imposed by hyaluronan accumulation and de novo three interacting loci, found on different chromosomes, in-
adipogenesis in GO. duce genetic susceptibility to GD (185). These data did not,
Proptosis, the forward displacement of the globe, stems however, support a major role for additional familial factors
from the increase in tissue volume within the unyielding in the development of severe GO in patients with GD (186).
confines of the bony orbit. Chemosis and periorbital edema These investigators tested four candidate genes, including
are caused primarily by decreased venous drainage from the HLA, TNF-, CTLA4, and TSHR, and found none to be
orbit, secondary to venous compression (170). Extraocular specifically associated with GO. Thus, it appears that envi-
muscle enlargement and dysfunction may result from GAG ronmental factors, rather than major genes, are likely to be
accumulation, edema, and inflammation in the endomysial the primary predisposing factors to the development of GO.
connective tissues and could lead to compressive optic neu-
ropathy and visual loss. In late stages of the disease, the
B. Smoking
extraocular muscles are sometimes fibrotic and atrophic, pre-
sumably as a result of chronic compression of muscle fibers. Although the prevalence of smokers among patients with
GD is higher than in controls, the relative risk of developing
GD in relation to smoking is quite small (odds ratio, 1.9; Ref.
VIII. Genetic and Environmental Contributions to 187). However, the association between smoking and GO has
GO Pathogenesis been shown to be much stronger, representing the strongest
A. Genetic contributions risk factor known for this condition. The odds ratio, relative
to controls, has been reported to be as high as 20.2 for current
GD is a complex, multigenic condition for which many of smokers and 8.9 for current and ex-smokers, suggesting a
the genetic contributions remain elusive. This disease ap- direct and immediate effect of smoking (188, 189). In addi-
pears to develop as a result of many interactions between tion, other studies have shown that among patients with GO,
relatively weak susceptibility genes and environmental trig- smokers have more severe eye disease than do nonsmokers
gers. Particular genes, including HLA (175178), CTLA4 (190).
FIG. 4. Lower extremities of a patient with

severe pretibial dermopathy.
The mechanisms involved in the association between smok- elevation of TSHR antibodies (196), and a transient increase
ing and GO are unclear. That smoking has been linked to other in serum pro- and antiinflammatory cytokines (197).
autoimmune diseases, including rheumatoid arthritis (191) and Several large prospective clinical trials have suggested a
Crohns disease (192), suggests that there may be a generalized direct relationship between 131I treatment and the develop-
stimulation of autoimmune processes in smokers. However, it ment or progression of GO (198, 199). The best designed of
is doubtful that changes in serum levels of cytokines are re- these studies examined hyperthyroid patients with mild or
sponsible, because concentrations do not differ in smokers and no GO who were initially treated with a 3- to 4-month course
nonsmokers, except that smokers do tend to have higher IL-6 of methimazole (200). Patients were then randomly assigned
receptor (IL-6R) levels (193). In addition, although patients with to receive radioiodine therapy, radioiodine plus prednisone,
hyperthyroidism and GO have higher levels of IL-6R receptor or continued methimazole therapy. Among the 150 patients
than those with hyperthyroidism alone (192, 194), IL-6R does treated with radioiodine, ophthalmopathy developed or
not appear to differ between smoking and nonsmoking GO worsened in 23 (15%) between 2 and 6 months after treatment
patients (193). These results would seem to suggest that ele- and improved in none. The worsened eye disease was tran-
vated IL-6R reflects, rather than causes, orbital disease. Thyroid sient and very mild in 15 of these patients and persistent in
hormone excess per se is unlikely to contribute to the association eight, seven of whom had evidence of GO before radioiodine
because other causes of hyperthyroidism or goiter, including treatment. Among the 145 patients treated with radioiodine
toxic nodular goiter, sporadic nontoxic goiter, and Hashimotos and prednisone, 67% had improvement, and none worsened.
thyroiditis, are not associated with smoking. Studies in vitro Of the 148 patients treated with methimazole, four patients
have demonstrated increased GAG production when orbital (3%) had worsening, whereas three patients improved. Al-
fibroblasts were cultured under hypoxic conditions (195). In though the frequency of worsened GO was significantly
addition, tobacco products enhanced IL-1 secretion by these higher at 6 months after treatment in the radioiodine group
cells, which in turn has been shown to increase GAG than in either of the two other groups (P 0.001), this effect
production. was very mild and was not present at 1 yr. The authors
concluded that worsening of GO after radioiodine therapy is
C. Radioiodine treatment for GD often transient (and mild) and can be prevented by the con-
current administration of prednisone to selected, high-risk
Recent interest has surfaced concerning whether the patients. Patients who might benefit from this duo-treatment
choice of therapy for hyperthyroidism in GD (131I ablation, include those with preexistent GO, smokers, and those having
surgery, or antithyroid drugs) has an effect on the develop- no significant contraindications to corticosteroid therapy (201).
ment or progression of GO. A causal relation between ra-
dioiodine treatment and the development of GO is plausible.
Such treatment could cause thyroid follicular disruption IX. Orbital Autoimmunity
with the release or new exposure of thyroid autoantigens, A. Immunohistochemical studies of orbital tissues
especially TSHR, although this has never been shown di-
rectly. Indirect evidence of this phenomenon occurring after A diffuse infiltration of lymphocytes, with sparse lym-
radioiodine treatment includes T cell activation, prolonged phoid aggregates, is present in the extraocular muscle inter-
stitial tissue and in the orbital fatty connective tissues of (Th2) immune response. Two groups of investigators re-
patients with GO (202). The majority of these cells are T ported that the majority of orbital T cell clones in GD produce
lymphocytes (CD2/CD3) and macrophages, with sparse B Th1-type cytokines IL-2, IFN-, and TNF-, but not IL-4 or
lymphocytes (Leu-26). Both helper/inducer (CD4) and IL-5 (207, 208). A third group detected the presence of mRNA
suppressor/cytotoxic (CD8) T lymphocytes are present, encoding a Th2 dominant profile IL-4, IL-5, and IL-10 (209),
with a slight predominance of the latter (203). A substantial whereas another group of investigators identified clones se-
proportion of the T cells, frequently adjacent to blood vessels, creting cytokines characteristic of subtypes IFN-, IL-4, and
are activated memory cells (CD3/CD45RO). IL-10 (210). These studies suggested that T helper cells of
Immunohistochemical studies have demonstrated the both subtypes may be represented in the retroocular infil-
presence of several cytokines, including IFN-, TNF-, and trates in GO, perhaps at different times during the course of
IL-1, within the retroocular tissues of patients with severe the disease. This shift in T cell repertoire could result from
GO (204). Immunoreactivity for these cytokines was noted epitope spreading within the same target antigen or involve-
both in the cytoplasm of infiltrating mononuclear cells and ment of different antigens. In the absence of information on
in the adjacent connective tissue. The preferential expression the antigen specificity of infiltrating T cells, it is very difficult
of these compounds in areas adjacent to mononuclear cell to fully evaluate the relevance of these findings to GO.
aggregates suggests that these cells are an important source In another study, a total of 117 T cell clones were estab-
of orbital cytokines. However, in the case of IL-1, marked lished from orbital adipose/connective tissues of six GO
immunoreactivity was also found in the fatty connective patients, and cytokine production was measured in 57
tissues remote from mononuclear cell infiltrates. Thus, it is CD3CD4 clones (211). Th1-Type clones were found to
possible that IL-1 is produced by infiltrating cells and fi- predominate in cultures from patients with recent onset (2
broblasts residing within the orbit. yr) of hyperthyroidism (Th1/Th0/Th2 57/29/14%) or GO
(Th1/Th0/Th2 47/30/23%). In contrast, Th2-type clones
B. Orbital T cell repertoire
predominated in cultures from patients with more remote
onset (2 yr) of hyperthyroidism (Th1/Th0/Th2 0/31/
A central role for T cells in GD and GO was suggested by 69%) or GO (n 4; Th1/Th0/Th2 0/25/75%). Although
several studies demonstrating restriction of TCR V region the CD3CD4 clones characterized were not necessarily
gene usage in Graves thyroid tissue or in orbital connective/ tissue-antigen-specific, these findings suggest that cell-
fatty tissues and pretibial tissue of patients with active, in- mediated (Th1-type) immune reactions may predominate in
flammatory GO (84, 85). The later study also found that the orbit in early, inflammatory stages of the disease. The
orbital tissues of patients with long-standing, inactive GO or proinflammatory cytokines (IFN-, IL-2, and TNF-) pro-
with unrelated orbital conditions showed minor or no re- duced by these cells, along with IL-1 derived primarily from
striction of TCR V gene usage (205). macrophages and fibroblasts, may prove responsible for
Recruitment of T cells with a wide range of specificity (i.e., stimulation of GAG synthesis in orbital fibroblasts and might
more divergent sets of expressed TCR genes) appears to function as mediators of inflammation in early disease. In
follow tissue destruction and cytokine-induced antigen ex- addition, the CD40/CD154 could serve as an important ac-
pression occurring later in the disease (206). In this study, tivational bridge between T cells and orbital fibroblasts.
comparison of TCR V gene usage between patients showed These same cytokines stimulate the expression of immuno-
clear heterogeneity. However, a few striking interpatient modulatory molecules (HLA-DR, ICAM-1, and HSP-72) on
similarities in V and V gene family usage were noted, orbital fibroblasts, potentially enhancing the propagation of
especially in V2 and V6 TCR genes. Taken together, these autoimmune responses within the orbit (166). In the recovery
observations indicate that a limited degree of clonality exists phase of GO, T helper type 2 lymphocytes (producing IL-4,
among certain populations of thyroidal, orbital, and pretibial IL-5, and IL-10) could play an important role and may me-
lymphocytes. These findings suggest that the antigen-driven diate the late-stage fibrosis of the extraocular muscles (212).
T cell response might be focused during the earlier stages of
the immune response in GO. However, later during the dis- D. Cytokine effects in the orbit
ease they exhibit heterogeneity in TCR gene usage. It may be
that the important antigen-specific T cells within the orbit Cytokines (213, 214) and chemokines (210) are capable of
undergo activation-induced apoptosis upon contact with the inducing the expression of immunomodulatory proteins in
relevant autoantigen and that the T cells remaining in the orbital fibroblasts and could contribute to the propagation of
orbit are nonspecific bystanders. the disease process. In addition, regulated on activation,
normal T cells expressed and secreted (RANTES), IL-16, and
C. Cytokine production in the orbit monocyte chemoattractant protein-1 may be important in
triggering T cell migration across activated microvascular
Most cytokines of pathological relevance in disease are endothelium into the orbit. IL-1, TGF-, IGF-I, IFN-, leu-
produced within the involved tissues. Therefore, measure- koregulin, and coculture with mast cells have been shown to
ment of serum cytokine levels may prove irrelevant in GO. stimulate accumulation of GAGs in orbital fibroblast cultures
Several studies have attempted to characterize the profile of (213217). Preliminary studies suggest that some cytokines
cytokines secreted by orbital-infiltrating T helper (CD4) can either inhibit (TGF-, IFN-, and TNF-) or stimulate
cells in GO to determine whether these cells are involved (IL-6) adipogenesis in cultures of orbital fibroblasts (218,
primarily in a cell-mediated (Th1) or a humoral-mediated 219). As will be discussed later in this review, enhanced
expression of the TSHR within the orbit may play a role in in sera from patients with GD, but with no distinct bias in
the initiation or propagation of the autoimmune response favor of the subgroup with GO, compared with 15% of con-
in GO. trols. Because neither the eye muscle membrane antigen nor
Cytokines are also capable of inducing the expression of the orbital fibroblast antigen was found to be restricted to
several proteins in orbital fibroblasts, including HLA-DR, orbital tissue and the antibodies were detected frequently in
intracellular adhesion molecule-1, and heat shock protein-72 normal individuals, it is not likely that these antibodies are
(166). If reflective of events occurring in vivo, their enhanced directly involved in GO pathogenesis.
expression in inflammatory states within the orbit would be The screening of a gt11 human thyroid cDNA library
expected to influence the ongoing autoimmune response. with a pool of Hashimotos thyroiditis sera led to the isola-
Another effect of cytokines relevant to GO is their ability to tion of a clone termed D1, encoding a 97-amino acid peptide
stimulate the proliferation of orbital fibroblasts. Significantly (228). The full-length cDNA was found to encode a 63- to
increased proliferation was observed after treatment with 64-kDa protein that was thought to be the same antigen as
IL-1, IL-4, IGF-I, and TGF-, but not with IL-2 or IL-6 (220). identified in earlier immunoblotting studies (229). However,
This effect of cytokines on orbital fibroblasts may be con- this could not be confirmed with peptide sequencing or an
tributory in the later stages of GO when fibrosis of the ex- analysis of amino acid composition. This protein was ex-
traocular muscles may result in debilitating double vision. pressed in thyroid and extraocular muscle, but not in skeletal
muscle. However, later studies, from a different group of
investigators demonstrated the presence of the D1 antigen
X. Orbital Autoantigens mRNA in a wide variety of tissues including uterus, spleen,
The close clinical associations and temporal features and parathyroid (230). Furthermore, antibodies to D1 were
shared by Graves hyperthyroidism and GO are reviewed found frequently in the sera of normal individuals, and their
above. These observations led to the hypothesis that GO is presence was not correlated with the presence of clinical GO
the result of an autoimmune response directed against one in Graves patients. Attempts to identify this antigen using
or more orbital autoantigens that are also present within the gel separation and microsequencing suggested that it might
thyroid. Such cross-reactivity might be a T cell function in- be either calsequestrin, a calcium-binding protein in the sar-
volving recognition of antigenic epitopes that have been coplasmic reticulum of striated muscle (231), or the flavopro-
processed by APCs within the orbit. Alternately, the cross- tein subunit of mitochondrial succinate dehydrogenase
reactivity could be primarily at the B cell level. In this case, (232). In subsequent studies by the same investigators, a
antibodies produced by activated B cells residing in the thy- novel protein, termed G2 s, was cloned from an eye muscle
roid, and likely also within the orbit, would specifically tar- gt11 library using an affinity-purified anti-55-kDa protein
get intact orbital cell-surface antigen. However, in contrast to antibody from a patient with GO (233). However, due in part
the well-documented occurrence of neonatal thyrotoxicosis to very low titers of relevant serum antibodies it proved very
due to transplacental transfer of anti-TSHR antibodies, the difficult to clone and identify a cDNA encoding this protein.
lack of any convincing reports of neonatal GO argues against Other investigators have concluded that these antigens rep-
the importance of orbital antibodies in pathogenesis. resent widely expressed cytoskeletal proteins. The inflam-
matory process occurring within the orbital tissues in GO
A. Target cells and candidate autoantigens
would lead to tissue destruction with release of these se-
questered proteins. Serum antibodies directed against these
Much effort has been directed at identifying target cells antigens would be unlikely to play a primary role in GO
and mapping the critical epitope on autoantigens they dis- pathogenesis.
play. The presence of gross muscle enlargement had led early Other candidate autoantigens studied in relation to GO
investigators to use eye muscle. In these studies, sera from include acetylcholine receptor and thyroglobulin. The
GO patients were used as probes in enzyme-linked immu- former was found in extraocular muscle membrane prepa-
nosorbent assays (221223). These studies involved crude rations to be recognized by antibodies from GO patients
preparations of soluble nonhuman eye muscle as the source (234). Subsequent studies, in which a human thyroid cDNA
of antigen, due to difficulties inherent in culturing muscle library was screened with polyclonal acetylcholinesterase
cells and a lack of available human eye tissue. Although the antibodies, led to the isolation of two thyroglobulin segments
presence of circulating antibodies directed against various having close homology with acetylcholinesterase (235).
eye muscle preparations was reported, the findings were not These findings were especially interesting in light of earlier
confirmed by other investigators or the antibodies were sub- studies in which thyroglobulin or a related protein was de-
sequently found to be nonspecific (224). Immunoblots dem- tected in the orbit by immunohistochemistry, and possible
onstrated serum reactivity against a 64-kDa eye muscle connections between the thyroid and orbit were shown using
membrane antigen (225, 226) and a 23-kDa orbital fibroblast radioisotope-based lymphography (236). Investigators hy-
antigen (227). The eye muscle membrane antigen was rec- pothesized that these connections might allow transfer of
ognized by approximately 70% of GO sera, especially those thyroglobulin into orbital tissues (237). However, the poten-
sera from patients with active or recent disease, and ap- tial significance of this finding is unclear, especially because
peared to react with a protein in the thyroid but not in patients with GO dont have particularly elevated antithy-
skeletal muscle. The 23-kDa protein was found to be cyto- roglobulin antibodies, and patients with Hashimotos thy-
solic, detected in cultured orbital fibroblasts from patients roiditis generally dont have evidence of GO. Weightman et
with GO; it was recognized by 56% of IgG class antibodies al. (238) showed that 125I-IGF-I binding to the surface of
orbital fibroblasts was displaced by IgG from patients with It is possible that TSHR is constitutively expressed at very
GD, suggesting that antibodies directed against the IGF-I low levels in extrathyroidal tissues in normal individuals,
receptor or IGF-I binding proteins might be present in the including orbital tissues, skin, and other areas not usually
serum. clinically involved in GD. This expression may be increased
in GD after stimulation by unknown factors, including par-
ticular inflammatory cytokines. Alternately, orbital TSHR
B. TSHR
expression may be enhanced secondarily as a consequence of
As discussed elsewhere in this review, the autoantigen orbital adipogenesis, which is increased in GO. In any event,
involved in the hyperthyroidism of GD is the TSHR. The increased local TSHR expression could make the receptor
close clinical relationships between GD, GO, and pretibial available to act as an autoantigen within the orbit, the
dermopathy make this protein a good candidate autoantigen pretibial skin, or other anatomic regions occasionally in-
for involvement in the orbital and dermal manifestations of volved in the disease. The presence of systemic subclinical
the disease. Several studies have examined the correlation connective tissue inflammation in GD, as has been suggested,
between the presence or level of TSHR antibodies and the might predispose to this series of events. Additionally, local
severity of GO with conflicting results. The most carefully factors, such as gravitational dependency, trauma, cigarette
designed of these studies found both thyroid stimulatory Igs smoking, or anatomic constraint of the bony orbit, may lead
(TSI) and TBII to be closely correlated with a GO clinical to clinical disease involvement at specific extrathyroidal sites
activity score (239). Weaker but significant correlation was (247).
also noted between antibody levels and proptosis. Several Evidence that orbital adipogenesis, with accompanying
studies, with conflicting results, have examined the relation- TSHR expression, may be involved in GO has been generated
ship between TSHR antibody titer and severity of GO. In GD, from cell culture studies of orbital preadipocyte fibroblasts
stimulatory TSHR antibodies cause hyperthyroidism. The (248, 249). These results obtained using human orbital fibro-
titers of these antibodies may reflect the severity of thyro- blasts are consistent with observations made in rodent fi-
toxicosis and degree of TSHR-specific lymphocytic activa- broblasts (250 252). Preadipocytes are cells of the fibroblast
tion (240). Similarly, because levels of TSHR antibodies may lineage that can, under certain culture conditions, differen-
reflect GO clinical activity, it may follow that infiltrating T tiate into adipocytes. Sorisky et al. (248) demonstrated that
cells found within the orbit in GO specifically recognize orbital fibroblasts contain a subpopulation of cells that be-
TSHR expressed in those tissues. The TSHR-directed anti- have like preadipocytes when incubated with cAMP-enhanc-
bodies produced subsequently, either locally or in the pe- ing compounds and prostacyclin. Initial studies disclosed
ripheral lymphatic tissues (i.e., lymph nodes and spleen), that 510% of the cells from a typical parental strain of orbital
however, may not have a direct pathogenic role within the fibroblasts undergo differentiation (248). Subsequent studies
orbit in GO but may rather reflect the intensity of the orbital from this same group have demonstrated that approximately
autoimmune response. However, whether TSHR or autoan- 50% of the cells undergo this differentiation when subjected
tibodies directed against it are involved in pathogenesis re- to medium-containing ligands of the adipogenic trigger, per-
mains uncertain. oxisome proliferator-activated receptor- (PPAR-) (249).
Studies aimed at determining whether TSHR is present in Moreover, the adipocytes fail to express surface Thy-1. Very
normal or GO orbital tissues or cell cultures led to reports by recent studies suggest that the subsets delineated in orbital
several laboratories identifying TSHR mRNA, or a variant fibroblasts by display of Thy-1 define discrete functional
TSHR transcript, in various orbital preparations using the populations of cells. Thy-1 cells fail to differentiate into
RT-PCR (241243). However, RNA transcripts detected only lipid-bearing adipocytes but when treated with TGF- de-
by PCR-based amplification of cDNA may have little phys- velop into myofibroblasts and express relatively high levels
iological relevance. To clarify this issue, further studies were of smooth muscle-specific actin (Koumas, L., T. J. Smith, and
performed using a ribonuclease protection assay for semi- R. P. Phipps, unpublished observations). In addition to un-
quantitative detection of this low abundance mRNA (244), dergoing adipogenesis, orbital cells show enhanced TSHR
Northern blotting (245) for mRNA detection, or immuno- expression when cultured under these conditions (249).
histochemistry (246) for TSHR protein detection. These stud- These findings suggest that there may be a humoral stimulus
ies documented the presence of mRNA and protein in GO for adipogenesis and TSHR expression present either sys-
orbital adipose/connective tissue specimens. In contrast, temically or within the orbit in GO. The cytokine IL-6 has
TSHR either was not detected or was found to be expressed been shown to enhance adipogenesis and TSHR expression
at low levels in normal orbital fatty connective tissue samples in cultured orbital preadipocyte fibroblasts (219). The cyto-
and derivative cultures. However, these studies failed to kine has been shown to be elevated in the sera of patients
examine samples of disease-derived and control tissues from with GD and GO (253, 254). The cellular source of IL-6 is
a large number of different donors under standardized con- uncertain. But it is of potential interest that IL-6 is expressed
ditions and therefore were probably not quantitative. In ad- by orbital fibroblasts and thyrocytes challenged with cyto-
dition, it should be noted that TSHR mRNA has subse- kines such as CD40 ligand (255257). It is possible that IL-6
quently been detected in a number of different tissues, serves as an important mediator of these changes occurring
including those not ordinarily involved in GD. Thus, addi- within the orbit in GO.
tional studies will be necessary to confirm that TSHR is a Studies concerning the pathogenesis of GD have long been
target autoantigen expressed at higher levels in orbital hampered by the lack of a useful animal model. Recently, a
tissues. novel animal model was developed in which thyroiditis was
transferred to naive BALB/c mice with splenocytes that were XI. Role of Orbital Connective Tissue in GO
primed with either TSHR fusion protein (136) or the cDNA
A. Orbital connective tissue is targeted by the immune
for the human TSHR (258). Thyroiditis was induced in the
system in GD
majority of these animals and was associated with the pro-
duction of low-titer TSHR antibodies, although the majority Why the connective tissue of the orbit and skin should be
of antibodies were TSH binding inhibiting, rather than stim- singled out for activation in GD is uncertain. One explanation
ulatory, Igs. Examination of the orbits in 17 of 25 of these concerns possible intrinsic differences in the residential or-
animals revealed lymphocytic and mast cell infiltration, ac- bital and leg cells as setting up these regions for disease
cumulation of adipose tissue and edema with material stain- involvement. Another concerns the potential for differences
ing with PAS, dissociation of muscle fibers, and evidence of in neurovascular investments, embryological derivation, and
TSHR immunoreactivity (257). Whether this particular ani- tissue architecture, promoting peculiar immunological reac-
mal model accurately reflects the autoimmune processes in- tivity. The impact of different local factors in sites of disease
volved in the development of GD is unclear. In fact, in an- involvement (247) cannot be excluded. Clearly, the vulner-
other study, vaccination with the naked TSHR elicited a Th1 ability to disease manifestations most likely reflects the
T cell response in which IFN-, rather than autoantibody, highly specialized function of the tissues. It is uncertain as to
production dominated the immune response (258). Although why only a small subset of patients with GD develop GO and
falling short of clearly exhibiting the spectrum of fully de- an even more limited population manifests clinically impor-
veloped GD or demonstrating a primary role for TSHR as an tant localized dermopathy, most typically in the skin of the
orbital target antigen, these models potentially open the anterior shin. A cell type common to both the orbit and shin
door for more robust models of the disease. is, among others, the fibroblast, a fundamental cellular build-
ing block of connective tissues. Fibroblasts represent the
most abundant cell type in these tissues, and their shared
C. Orbital T cell reactivity attributes could potentially tie together the two regions.
These cells represent the precursor from which other, more
Direct evidence that any particular candidate antigen or specialized cells develop (chondrocytes, adipocytes, and
orbital cell type is an autoimmune target in GO has been myofibroblasts are examples). It thus seems reasonable to
difficult to obtain, largely due to difficulties in obtaining focus inquiry on the peculiarities of the fibroblast phenotype
orbital lymphocytes and autologous orbital tissues from GO as underlying the characteristic tissue remodeling found in
patients. However, several studies have examined reactivity GD. Indeed, a number of studies have appeared in the lit-
of orbital T cell lines against various purified antigens or erature over the past few decades examining the behavior of
orbital tissue preparations. In one report, CD8 T cell lines orbital and pretibial fibroblasts in culture and, in some cases,
recognized autologous cultures of orbital fibroblasts in an contrasting them with those harvested from other anatomic
MHC class I-restricted manner (259). These T cells also pro- regions. These fibroblasts exhibit potentially important dif-
liferated in response to thyroid membrane preparations and ferences in their phenotypes that we believe account for the
purified TSHR. No proliferation in response to crude eye susceptibility of the orbit and lower leg to manifestations of
muscle extract, autologous peripheral blood mononuclear GD. The complex interplay between immunocompetent cells
cells, allogeneic fibroblasts, or purified protein derivative of and fibroblasts is summarized in the schematic contained in
mycobacterium tuberculosis was noted. Another group of Fig. 5.
investigators found that orbital and peripheral T cell lines The early molecular and cellular events associated with
from patients with GO proliferated in response to autologous GO and localized dermopathy are uncertain but presumed
orbital fibroblast-derived proteins in the 6- to 10-kDa and 19- to be proximately related. This assumption derives from the
to 26-kDa range but did not recognize proteins derived from fact that virtually all patients who develop dermopathy also
autologous orbital myoblasts or abdominal adipose or mus- have severe GO. Thus, dermopathic patients essentially rep-
cle tissue (260). Candidate antigens, including recombinant resent a subset of those with orbital manifestations. Because
D1 and thyroglobulin, were tested in another set of experi- the vast majority of patients with GO fail to manifest clini-
ments (261). Although no T cell responses to these antigens cally important skin disease, an additional provoking factor
were observed, cells from some patients exhibited weak re- might be necessary for the latter to appear. The tissue re-
sponse to eye muscle proteins between 25 and 50 kDa. In modeling associated with both processes seems, at least su-
contrast, orbital fibroblasts induced striking proliferation of perficially, to be related. As will be discussed, accumulation
circulating T cells from some patients. of the abundant GAG hyaluronan features prominently in
The interpretation of these studies is limited, in part, by the tissues affected by both processes.
their reliance on peripheral T cells or on orbital T cell lines,
rather than cloned T cells derived from affected orbits. In
addition, the use of whole or fractionated cells, rather than XII. Role of GAGs in GO
recombinant proteins, as the antigens precluded precise an-
A. GAGs accumulate in GO and dermopathy
tigen identification. Studies of endogenously processed and
presented antigens would be particularly useful in this re- Orbital and dermal lesions from patients with GD have
gard. Such studies would no doubt facilitate an understand- accumulations of material that assumes a metachromatic
ing of immune responses involved in the development quality when stained with either alcian blue or toluidine blue
of GO. (262). In 1942, this material was characterized by Trotter and
FIG. 5. Schematic diagram of a proposed

model for interactions between orbital fi-
broblasts and immunocompetent cells.
The complex molecular events are cur-
rently thought to drive cell activation and
tissue remodeling. PLC, Phospholipase
C; PKC, protein kinase C; TRE, thyroid
hormone response elements; G, G pro-
tein; GPCR, G protein-coupled receptor;
TSI, thyroid stimulatory Igs; HA, hyalu-
ronan; EP2, PGE-2 receptor type 2.
[Adapted from T. J. Smith: Curr Opin
Endocrinol Diabetes 9:393 400, 2002
(345).]
Eden (263) who found that the accumulation of mucin was Among the distinct molecular characteristics displayed by
common to both generalized (hypothyroid) and localized GAGs are rheological properties that underlie their ex-
myxedema associated with GD. The substance has proved at tremely hydrophilic nature and their viscosity in solution.
least partially susceptible to digestion with testicular hyal- Hyaluronan differs from the other abundant GAGs in that it
uronidase, suggesting that it is largely comprised of hyalu- does not contain a core protein and it is not sulfated, unlike
ronan. In dermopathic lesions, it would appear that chon- chondroitin, dermatan, and heparan sulfates. It would ap-
droitin sulfate levels may be decreased and those of pear that of these molecules, hyaluronan is accumulating
dermatan sulfate unchanged. On the other hand, Watson and predominately in connective tissue lesions of GD. It is com-
Pearce (264) found that both hyaluronan and chondroitin posed of alternating residues of N-acetylglucosamine and
sulfate content were increased substantially. The early, pio- d-glucuronic acid (262). The repeating sugars form a basic
neering work of Smelser, Asboe-Hanson, and others (265, disaccharide organization with alternating linkages. Hyalu-
266) provided the first insights into the chemical nature of ronan synthesis occurs at the plasma membrane where the
tissue remodeling in GD. It became apparent quite early that alternating sugar residues are transferred sequentially from
the water-binding properties of orbital tissues change as a their respective uridine diphosphate (UDP) donors by hy-
consequence of GO (267). Moreover, the changes were sim- aluronate synthases (270) (Fig. 6). This enzyme activity lo-
ilar to those found in generalized myxedema associated with calizes to the membrane fraction of disrupted oligodendro-
hypothyroidism (263). Asboe-Hansen and Iversen (268) and glioma cells in culture. Great progress in defining the
Ludwig et al. (269) demonstrated that ground substance ac- molecular biology of hyaluronan synthesis has been made
cumulation was increased in experimentally induced GO in over the past few years. A family consisting of three hyalu-
guinea pig and that it comprised, in large part, hyaluronan. ronan synthase (HAS) isoforms has been identified recently,
Wegelius et al. (266) reported one of the earliest analyses of and all members have been cloned (271273). The tissue
human orbital contents in GO. They found that numerous distribution of the HAS enzymes appears to differ, as does
well-granulated mast cells and lymphocytes populated the the putative role that each might play in normal development
tissues, which appeared metachromatic when stained with and in the pathogenesis of human disease. The expression of
toluidine blue. This material was present in both connective all three HAS transcripts has been shown to be up-regulated
tissues and in the muscle. It proved on further inspection to by IL-1 in cultured human orbital fibroblasts (219). In ad-
represent mucopolysaccharides, a previously used term for dition, the enzyme immediately upstream from the HAS
GAGs. enzymes, termed UDP-glucose dehydrogenase (UDP-GD),
Hyaluronan is a high molecular weight molecule and is a has also been characterized (274). This enzyme catalyzes the
member of a group of complex carbohydrate molecules conversion of UDP-glucose to UDP-glucuronate. UDP-GD is
called GAGs (262). GAGs are linear polymers with charac- induced substantially by proinflammatory cytokines in or-
teristic physicochemical properties. They are large, bulky, bital fibroblasts, and this induction can be attenuated by
polyanionic compounds composed of repeating disacchar- physiologically relevant concentrations of glucocorticoids
ides, each of which comprises an amino sugar and a uronic (274). It is unclear whether, in the case of GD, hyaluronan
acid residue. One molecule of hyaluronan with a molecular synthesis is accelerated or whether macromolecular disposal
weight of 106 Da possesses a spherical diameter of 4,000 might be delayed. Hyaluronan degradation is catalyzed by
and a volume of 330,000 1019 ml. An equivalent weight a group of enzymes called hyaluronidases. Depending on
of hyaluronan occupies a volume that is 75,000 times greater their tissues of origin, they exhibit distinct profiles of sub-
than that of collagen. When covalently linked to core pro- strate specificity. Bacterial hyaluronidases, such as those ex-
teins, complexes of GAG are known as proteoglycans. pressed by Streptomyces, are extremely specific for hyaluro-
FIG. 6. The biosynthesis of the GAG, hyaluronan

involves multiple enzymes.
nan (275). They are therefore particularly important tools for the cell surface and is competent to signal downstream
the biochemical analysis of GAGs. Whereas animal fibro- events through the activation of kinase cascades (280). An-
blasts express hyaluronidases, those from human skin and other hyaluronan binding protein/receptor of potential im-
orbit fail to produce hyaluronan degrading enzymes (276, portance is RHAMM (281). This protein, first described by
277). These differences in hyaluronidase expression found in Turley and colleagues (281), binds with high affinity to hya-
fibroblasts from different sources may reflect interspecies luronan. That group contends that hyaluronan binds to
variations or may indicate loss of a particular phenotypic RHAMM and in so doing stimulates cell locomotion. More-
attribute in human fibroblasts. Although GAGs function in over, this effect may be mediated through rapid and transient
the extracellular compartment, they may need to be trans- protein tyrosine kinase signaling (281). Although the func-
ported into lysosomes for complete degradation. Human tion of this second receptor is less well-defined than that of
skin fibroblasts are incapable of internalization of hyaluro- CD44, binding of hyaluronan to target cells through
nan (278), unlike other cell types, and this may explain their RHAMM may lead to changes in cell biology and gene ex-
inability to degrade the macromolecule completely. Because pression (282). Emerging as an important direction for future
human fibroblasts do not appear capable of degrading hya- investigation is a clearer definition of the molecular events
luronan, it is essential that other cell types might express underlying hyaluronan accumulation and the biological con-
hyaluronidases in vivo, the human orbit. Alternatively, hya- sequences of a disordered economy of the complex sugar
luronan disposal could occur through relatively nonspecific in GO.
pathways.
Whereas the disordered accumulation of GAGs in some B. Orbital fat expansion and muscle enlargement can
tissues might fail to provoke serious tissue dysfunction, in contribute substantially to tissue remodeling in GO
the orbit, such an infiltration of hydrophilic macromolecules
is frequently associated with profound mechanical conse- Although most individuals with GO present with evi-
quences. This results from the tight and unforgiving spatial dence of both muscle enlargement and increased volume of
constraints imposed by the bony walls of the orbit. The the fatty depot in the orbit, some patients appear to exhibit
increase in soft tissue volume associated with GO can result a predominance of either (283). Those individuals younger
in the partial expulsion of the globe, a condition termed than 40 yr are considerably more likely to manifest orbital fat
proptosis. Depending on the magnitude of this tissue ex- expansion-related proptosis in the absence of muscle infil-
pansion and the degree of eye displacement, neurovascular tration (283), whereas older patients, more than 70 yr old, can
embarrassment and vision loss might occur. In addition, the develop severe, isolated muscle enlargement associated with
inability to fully close the eyelids could lead to severe and compressive optic neuropathy (284). This presentation can be
sight-threatening ulceration of the cornea. attributed to fusiform enlargement of the extraocular mus-
Many of the consequences of hyaluronan accumulation in cles. When assessed early in the course of the disease, the
the orbit result from material bulk. In addition, low molec- contractile elements of the muscle remain intact in those few
ular weight hyaluronan has been shown to influence gene patients in whom such an examination has been possible. It
expression of cells exposed to the complex carbohydrate would appear that the connective tissue investments sur-
(279). In particular, the potential for hyaluronan to induce rounding the muscle itself are altered (285). Specifically,
proinflammatory genes in immunocompetent cells could there is accumulation of GAGs and infiltration of the per-
lead to substantial amplification of the inflammatory re- imysium with mononuclear cells, including T lymphocytes
sponse. Many of the actions of hyaluronan on target cells are and mast cells.
mediated through its binding to CD44, which is displayed on Orbital fat has been the topic of investigation for many
years. The elegant, pioneering work of Smelser examined the the histological picture of late GO, their prominence in many
fat of animals in which experimental ophthalmopathy had examples of dermopathic skin is less obvious. In addition to
been induced by first thyroidectomizing guinea pigs and lymphocytes, mast cells are often abundant (174). This may
then injecting the animals with extract of the anterior pitu- have substantial importance because of the role that these
itary (265). The pituitary extract produced a 35% increase in cells play in the genesis of fibrosis. Both T cell phenotypes are
the weight of the orbital contents. Only the ventral lacrimal present in orbital lesions late in the disease (258, 289, 290), but
gland was spared. The fat of the orbit nearly doubled in whether CD4 or CD8 cells are dominant early remains
weight and acquired a different appearance; it became ge- controversial. A number of cytokines have been detected in
latinous and translucent rather than opaque as in the control the orbital tissues in GO. Among these are TNF-, IL-1,
animals. Edematous fluid appeared to have infiltrated be- IFN-, and TGF- (207). These studies have relied largely on
tween fat cells. The edematous material stained strongly with RT-PCR-based assays attempting to quantify cytokine
aniline blue or eosin but failed to stain like amyloid or mu- mRNAs, or they used immunostaining. Although the reports
coid material. have provided the first approximation of the cytokine milieu
Orbital fat has been examined in the context of GO using found in GO, they are neither specific nor quantitative. More-
multiple imaging modalities. Measurement abnormalities over, important and direct comparisons between the cyto-
were found in 87% of patients with GD with clinically de- kine profiles found in GO and other types of orbital inflam-
tectable GO. In contrast, 70% of hyperthyroid patients with- matory diseases have yet to be conducted. These and more
out clinical signs were found to have abnormal pixel-cali- quantitative studies will be necessary to define those mo-
brated volume measurements of muscle and fat by CT scans lecular triggers present in the earliest stages of pathology that
in a study involving 72 subjects (171). Enlargement of fat in might distinguish GO from other forms of orbital inflam-
addition to muscle was documented in 46% of patients and mation. The lack of robust animal models for GO has also
isolated volume expansion of fat in 8%. In another study, 15 proven an important limitation in defining the cellular and
patients presenting with proptosis but without masses or biochemical events that provoke initiating events. The orbital
muscle enlargement were studied, and four were found to manifestations contained in most reports of experimentally
have GO (285). induced rodent disease have been disappointing (257, 291).
The mechanism involved in fat enlargement in GO is not
understood but may reflect a shift in the levels and local
impact of adipogenic factors to which the orbital tissue is XIII. Role of Orbital Fibroblasts in GO
exposed. In general, it has become evident that adipogenesis A. The putative role of orbital fibroblasts in the
is a tightly regulated process and that key molecules initiate pathogenesis of GO
the differentiation of preadipocytes to mature fat cells. Can-
didate molecules include natural ligands of PPAR- (286, Human fibroblasts were once viewed as relatively inert,
287). The best characterized of these are prostaglandins (PGs) nonreactive cells engaged almost exclusively in housekeep-
of the PGJ2 series and related prostanoids. Unfortunately, no ing structural activities. They were thought to participate
information currently exists concerning PG production or mainly in the elaboration and organization of extracellular
tissue levels in orbital tissue in situ. Moreover, whether GO matrix (ECM), participating in its disposal and directing the
is accompanied at least in some patients with a shift in the assembly of the pericellular microenvironment in which
homeostatic mechanism governing orbital fat mass is not more specialized cells might function. This perception has
known. Of potential relevance to the issue of enhanced adi- begun to change, and we now more fully appreciate the
pogenic differentiation in GO is the finding of fatty infiltra- complex array of activities in which fibroblasts appear to
tion in extraocular muscle (280). Although no stringent quan- participate (292, 293). They both respond to and produce
titative analysis of hyaluronan content in orbital tissues numerous molecular signals that serve to activate and mod-
currently exists, several qualitative observations suggest that ulate the behavior of bone marrow cells and provoke their
the GAGs accumulate in both muscle and connective tissues. migration toward sites of inflammation (294). Thus, fibro-
Thus, the increase in fat cell number and or size may con- blasts may initiate some of the very earliest events that lead
tribute to fat expansion in GO (288). But the contribution of to an inflammatory response. Moreover, they synchronize
infiltrating GAGs to this process has not been quantified. the evolution of tissue remodeling and, when chronically
Much of the insight into cellular infiltration of the orbital activated, direct the process of fibrosis. In this regard, they
tissues in GO derives from histological examination rela- may function as sentinels involved in transducing danger
tively late in the course of the disease. The paucity of infor- signals and allowing them to culminate in mature immune
mation causing early GO results from a lack of access to responses. Fibroblasts are capable of sounding an alarm to
tissues before the end stages of the disease. There exist few alert the cellular neighborhood of stressful tissue events.
indications for surgical intervention during the active By virtue of their elaboration of chemoattractant molecules
phases. Consequently, we do not know the identity of the such as chemokines, they provoke the trafficking of bone
initial immunocompetent cell types recruited to the orbit that marrow-derived cells to sites of inflammation (294). The
share proximate responsibility for the earliest processes. profile of small molecules emanating from activated fibro-
These cells orchestrate the inflammatory response and ini- blasts is believed to exert a substantial bias concerning the
tiate tissue remodeling. Rather, the relatively few reports phenotypes of bone marrow-derived cells that infiltrate tis-
concerning infiltrating cells in GO appear to define the re- sues during the initial stages of inflammation. The remaining
active components of the disease. Although T cells dominate question concerning which attributes define unique fibro-
blast properties has not been answered. Fibroblasts exhibit a behavior in synovial cultures is relevant to those from orbit,
distinctive morphology. To date, no universally accepted one would expect similar profiles of PGE2 production in
marker has been shown definitively specific to fibroblasts, those cells.
and the identification of fibroblasts rests largely on exclu-
sionary criteria. Cells recognized as fibroblasts are generally
B. Orbital fibroblasts are different from many other types
described as stellate to angular with several cytoplasmic
of fibroblasts
processes. They are known to migrate on substratum and can
become deformed when subjected to osmotic stress. More- Human fibroblasts have been examined rather extensively
over, they may be thought of as precursor cells that give rise in culture in an effort to understand their potential role in the
to more specialized components of connective tissue, includ- pathogenesis of GD. In particular, cultures derived from the
ing exhibiting the contractile properties of myofibroblasts skin of the anterior leg and the orbits have gained substantial
and undergoing adipogenesis. attention, largely because the lesions affecting connective
Recent evidence has been advanced to support the concept tissue in GD occur most frequently in those areas. Among the
of fibroblast diversity. Fibroblasts from certain anatomic re- first studies involving cultured orbital fibroblasts were those
gions appear to differ from those found elsewhere in the of Sisson and colleagues (303305). Sisson characterized the
human body. Moreover, within certain tissues, subpopula- GAG synthesis in orbital fibroblast cultures derived from the
tions of cells can be identified. Discrete subsets of fibroblasts connective tissues. Those studies revealed that orbital fibro-
can now be identified on the basis of expression of cellular blasts exhibited a substantial response to lymphocytes with
markers such as surface proteins, carbohydrates, and gan- regard to hyaluronan synthesis glucose utilization (304). Sis-
gliosides. Some of those subpopulations appear to represent son and Vanderburg reported that glucocorticoids failed to
functional subsets. Identification of cells within a tissue that inhibit basal hyaluronan synthesis in orbital fibroblasts but
behave differently suggests that the physiological roles of could block lymphocyte-provoked GAG synthesis in those
subsets in health and disease may diverge. This phenomenon same cells (305). A more recent report demonstrated a similar
may resemble the situation concerning lymphocytes, where relative insensitivity of hyaluronan synthesis in orbital fi-
cells are distinguished on the basis of surface determinants broblasts to the actions of T3 and dexamethasone (277). Hya-
and marked functional differences among them have luronan synthesis in dermal fibroblasts has been shown to be
emerged. Thus, the realization that discrete fibroblast subsets inhibited substantially by physiological concentrations of
populate some tissues represents an important insight into glucocorticoids (306) and thyroid hormones (307). Whether
the complexity of tissue organization. The pioneering obser- orbital fibroblasts are insensitive to these hormones as a
vations concerning fibroblast subsets within a tissue were consequence of differences in receptor complement or post-
made by Phipps and his colleagues (293). They discovered receptor signaling is uncertain.
that some fibroblasts from the murine lung fail to express and Orbital fibroblasts, like those derived from certain other
display surface Thy-1. This determinant represents a surface anatomic regions, exhibit phenotypic attributes that set them
glycoprotein, the natural ligand of which has yet to be iden- apart. They have a distinct morphology in vitro (277). Orbital
tified (294). Thy-1 has been shown recently to bind integrin fibroblast cultures contain both angular (stellate-shaped with
3 on astrocytes and, in so doing, to trigger the tyrosine three or more cytoplasmic processes) and fusiform (spindle-
phosphorylation of focal contacts (295). Phipps et al. (296) shaped with two or three dendritic processes) cells (277). The
determined that the surface display of Thy-1 by murine lung perinuclear cytoplasm contains prominent granular features.
fibroblasts defined cells with several phenotypic attributes Dermal fibroblasts examined at the same time contain pre-
that appeared divergent. For instance, only Thy-1 fibro- dominantly angular cells with a more homogeneous and less
blasts display class II major histocompatibility complex an- granular cytoplasm. In contrast, the ultrastructure of orbital
tigens after stimulation with IFN- (296). When activated and nonorbital fibroblasts is remarkably similar, as deter-
with TNF, only the Thy-1 fibroblast subset expresses IL-1 mined by transmission electron microscopy (308). Orbital
(297). In contrast, Thy-1 fibroblasts failed to express either fibroblasts display receptors (309) and gangliosides (310) and
IL-1 or IL-1. Thy-1 fibroblast subsets produce substan- generate macromolecular components of the ECM differ-
tially more collagen in vitro, suggesting that these cells might ently than do other fibroblasts (214, 311). Using two-dimen-
have particular importance in the pathogenesis of fibrosis sional protein gel electrophoresis, Young et al. (312) have
(298). On the other hand, IL-6 appears to be expressed in both systematically compared the basal and cytokine-provoked
Thy-1 and Thy-1 subsets (299). Moreover, the expression protein expression in orbital fibroblasts and compared this
of immunologically important molecules, such as PGs, ap- profile with those of the pretibial and abdominal wall fibro-
pears to differ in the two fibroblast subsets. Thus, precedent blasts. Notable in these studies was their use of multiple
exists for subsets exhibiting very distinct phenotypic char- strains from a single donor. They found that, although the
acteristics that would be potentially important for how they vast majority of protein spots were expressed at similar levels
might participate in inflammatory responses and fibrosis. of abundance in all three sites and responded the same way
Korn and his colleagues (300 302) have demonstrated sub- to cytokine treatment, a limited number of protein inductions
stantial clonal variations with regard to PGE2 synthesis and repressions were restricted to orbital and/or pretibial
among synovial fibroblasts. These differences among subsets fibroblast strains (312). The substantial differences between
were stably expressed through many population doublings orbital and nonorbital fibroblasts suggest that the former
and substantial time in culture. Moreover, they were not may play unusual if not unique roles in the normal function
restricted to a particular stimulus. If the pattern of fibroblast of the orbit. Plasminogen activator inhibitor type 1 is a key
regulator of the pericellular proteolytic environment. When could represent the cells involved in fibrosis and respond to
orbital fibroblasts are treated with IFN- (313) or leukoregu- Th2 cytokines, including IL-4, IL-5, and IL-13. These cyto-
lin (314), plasminogen activator inhibitor type 1 is dramat- kines are associated with robust fibrotic tissue responses. The
ically induced. In contrast, expression in dermal fibroblasts increases in muscle and fat volumes associated with GO
is either down-regulated or modestly enhanced. This sug- could, at least in part, result as a consequence of tissue ex-
gests that in the context of an inflammatory response, orbital pansion from additional muscle and adipocytes differenti-
fibroblasts exhibit a differentially attenuated proteolytic en- ating from the respective fibroblast compartments. These
vironment that would favor the accumulation of ECM mac- apparent differences between fibroblasts from the muscle
romolecules. This difference may underlie some of the qual- and fat depots might help explain the diverging clinical pre-
itative tissue differences found in GO. sentation seen in old and young patients with GO (318).
It would appear that the population of orbital fibroblasts
can be further separated on the basis of specific phenotypic
C. Orbital fibroblasts express high levels of inducible
attributes. For instance, approximately 50% of parental
cyclooxygenase and produce extremely high levels of PGE2
strains of orbital fibroblasts display Thy-1 on their cell sur-
when activated by proinflammatory cytokines
faces (315). Smith et al. (316) reported that separating cells
into pure Thy-1 and Thy-1 cultures led to subsets that Orbital fibroblasts exhibit exaggerated responses to proin-
faithfully maintained their status over many population dou- flammatory signals such as those conveyed by cytokines. For
blings and passages in vitro. When segregated, Thy-1 cells instance, leukoregulin, a T cell-derived cytokine, and IL-1
produce more PGE2 and express higher levels of PG endo- dramatically up-regulate several genes that are relevant to
peroxide H synthase (PGHS)-2 upon treatment with IL-1 or inflammation. These include PGHS-2 (EC 1.14.99.1), the in-
CD154 (317). On the other hand, Thy-1 fibroblasts express flammatory cyclooxygenase (319, 320). When PGHS-2 is in-
much higher levels of IL-8 when activated with proinflam- duced in orbital fibroblasts derived from either normal or GO
matory cytokines (317). Parental strains of orbital fibroblasts connective tissue, a dramatic increase in PGE2 synthesis oc-
contain preadipocytes that, when subjected to a differenti- curs (319). The magnitude of this increase, provoked by a
ation protocol, undergo terminal differentiation into fat cells number of cytokines, is considerably greater than that ob-
(248). The medium prompting adipogenesis contained served in dermal fibroblasts (319, 320). The disparity in levels
cPGI2, insulin, dexamethasone, TSH, and isobutylmethyl- of PGHS-2 induction may relate to the relatively modest
xanthone (312). Approximately 5% of the cells differentiate levels of IL-1 receptor antagonist expression observed in
into cells that accumulate inclusions staining positively with cytokine-activated orbital fibroblasts (320). The levels of
Oil Red O. It was subsequently demonstrated that Thy-1 PGHS-2 mRNA and protein achieved after cytokine treat-
subset exhibits substantial adipogenic potential and can dif- ment are proportionately greater than those in other fibro-
ferentiate in vitro into mature adipocytes when incubated blasts, and the vast majority of the increased PGE2 produc-
with ligands of PPAR- (316). Such an adipogenic differen- tion observed is inhibited by PGHS-2 selective inhibitors
tiation in orbital fibroblast cultures has been associated with such as SC58125 (319). This finding suggests that PGHS-2
increases in the levels of expression of the TSHR (249). Un- selective inhibitors, now in the marketplace, might represent
fortunately, those reports contained data that were not con- effective and safe alternative therapeutics for acute orbital
vincing. The magnitude of the effects ascribed to differenti- inflammation in GO.
ation with regard to TSH-provoked cAMP generation was The induction of PGHS-2 requires the activation of the
inconsistent, and the usual indications of result variability MAPK pathways, including both ERK 1/2 and p38 (321).
were missing in several studies. Thy-1 cultures may not Moreover, nuclear factor (NF)-B, a centrally important tran-
possess the potential to differentiate into fat cells. The dem- scription factor in inflammation-related gene induction, ap-
onstration of adipogenic potential in orbital fibroblasts has pears critical to the activation of the PGHS-2 promoter in
substantial implications concerning the pathogenesis of GO. orbital fibroblasts (320). Thus, it would appear that the ex-
Expansion of orbital fat is a prominent feature of the disease aggerated induction of PGHS-2 in orbital fibroblasts helps
associated in some patients with GO (284). Clearly, the full define the inflammatory phenotype of these cells. In contrast,
spectrum of cellular differences imposed by the bimodal PGHS-1 are a constitutively expressed enzyme that is abun-
distribution of Thy-1 on orbital fibroblasts from connective/ dant in unprovoked fibroblasts (319). Moreover, leukoregu-
adipose tissue has yet to be appreciated but could define lin and IL-1 fail to alter the levels of PGHS-1 mRNA or
discrete functional characteristics of each subset. In contrast, protein. Although the expression and cytokine induction of
fibroblasts associated with and derived from the extraocular PGHS-2 are completely attenuated by physiological concen-
muscles display Thy-1 uniformly (317). Like dermal fibro- trations of glucocorticoids, PGHS-1 levels are unaltered by
blasts, those from the perimysial connective tissue are resis- the steroids.
tant to these differentiation protocols (317). This raises the Another enzyme in the PGE2 pathway is also induced by
possibility that Thy-1 fibroblasts can be terminally differ- proinflammatory cytokines in orbital fibroblasts (321). Mi-
entiated into other types of cells such as myofibroblasts. A crosomal PGE2 synthase (mPGES; EC 5.3.99.3) is the terminal
number of reports have appeared to suggest that fibroblasts catalytic determinant in the synthetic cascade for PGE2.
exposed to TGF- can be induced to differentiate into cells mPGES is a glutathione-dependent enzyme that is usually
possessing a phenotype associated with myofibroblasts. expressed at low levels under unstressed cellular conditions.
These cells express high levels of smooth muscle-specific mPGES is substantially induced by IL-1 in these cells in a
actin. In this regard, fibroblasts lacking adipogenic potential well-coordinated manner with PGHS-2. Induction of the two
enzymes by IL-1 shares utilization of the MAPK pathways matory bowel disease (329), and lupus erythematosis (330).
and involves the modest up-regulation of respective gene The other abundant chemoattractant synthesized by cyto-
promoter activities. PGHS-2 mRNA is extraordinarily labile kine-activated fibroblasts is RANTES, a c-c type chemokine
under basal culture conditions in human orbital fibroblasts (331). Induction of RANTES usually occurs at a pretransla-
due to the presence of at least 22 AUUUA instability elements tional level. In T cells, RANTES engagement of the cytokine
in its 3 untranslated region. When cells are treated with receptor CCR5 results in Janus kinase kinase and p38 MAPK
IL-1, the stability of PGHS-2 mRNA is dramatically en- activation, which in turn leads to downstream target gene
hanced. The half-life increases from 1 h to greater than 5 h. activity (332). RANTES has also been implicated in human
This augmentation in the half-life of the PGHS-2 transcript autoimmunity. Moreover, it has been detected in the thyroid
is critical to the up-regulation of the expression of the england of patients with GD (333).
zymes. In contrast, mPGES mRNA is extremely long-lived The mechanisms through which chemokines and related
under basal conditions, and IL-1 does not influence its molecules alter the migration of immunocompetent cells re-
survival appreciably (321). We hypothesize that the capacity main uncertain but probably relate to complex interactions
of orbital fibroblasts to produce PGE2 is the basis, at least in with these cells. The notion that lymphocytes and other tar-
part, for the susceptibility of the orbit to inflammatory pro- get cells might discriminate a concentration gradient across
cesses and the tissue remodeling seen in GO. PGE2 exerts an their diameters and adjust their movement toward higher
important influence on the nature of immune responses. For concentrations is probably incorrect. But regardless of the
instance, the prostanoid biases the commitment of naive T mechanisms involved in their actions, these small molecules
cells Th0 away from the Th1 phenotype and toward that of are of substantial importance in defining cell movements and
the Th2 (322). PGE2 plays an important role in the behavior infiltration of tissues, such as those targeted in GD.
and apoptosis of B lymphocytes (323) and influences mast Human fibroblasts express high levels of IL-16 mRNA
cell function (324). Thus, the finding that orbital fibroblasts under basal conditions but not pro-IL-16 or mature IL-16
generate particularly high levels of PGE2 when activated protein. In contrast, RANTES mRNA is undetectable in these
suggests the potential of those cells in defining the quality of cells under control conditions. When the fibroblasts are
tissue remodeling found in inflammatory disease of the orbit, treated with proinflammatory cytokines, such as IL-1,
such as occurs in GO. The result of this bias can substantially TNF-, TNF-, or leukoregulin, they express high levels of
skew the cytokine microenvironment toward profibrotic fac- mature IL-16 and RANTES proteins that are released from
tors and away from those associated with acute inflamma- the cell layers. Moreover, both proteins are active and com-
tion (325). petent to initiate T cell migration (334). Inhibitors of
caspase-3 activity block the release of IL-16 from fibroblasts
D. Fibroblasts are sentinel cells capable of initiating
but fail to influence RANTES expression. Glucocorticoids
lymphocyte recruitment and tissue remodeling
block the expression of both molecules. Greater than 90% of
the T cell migratory activity generated by cytokine-treated
Fibroblasts from a wide array of human tissues have been fibroblasts can be attributed to IL-16 or RANTES, suggesting
shown to express chemoattractant activities when activated that these two chemoattractants represent predominant mo-
by cytokines. The role of recruiting leukocytes to areas of lecular triggers, emanating from fibroblasts that orchestrate
inflammation and tissue damage falls, in some measure, to lymphocytic infiltration at sites of tissue injury and repair.
a family of cytokines called chemokines. These are small If cytokine-activated fibroblasts from many tissues express
polypeptides that range in size from 7 to 10 kDa and com- high levels of IL-16, RANTES, and other important chemoat-
prise different families, depending on their amino acid se- tractant molecules, it is unclear why the manifestations of GD
quences. They are designated according to the cysteine res- are not more generalized. One possible explanation concerns
idue signatures they contain and are divided into three a global infiltration of immunocompetent cells to many tis-
families. Chemokines bind to high-affinity receptors ex- sues and the differential impact those cells might have on the
pressed on the surfaces of target cells. The various members infiltrated tissues. That model would be consistent with lym-
of the three families exhibit diverse specificity with regard to phocyte-derived activating molecules exerting site-depen-
the receptors they utilize and the target cells they activate. In dent effects on fibroblasts and other residential cells in a
addition to chemokines, fibroblasts express other small mol- particular anatomic region. From the body of evidence thus
ecules that lack requisite cysteine signatures but possess far advanced, it would appear that orbital fibroblasts are
activities that prompt leukocytes to infiltrate areas of tissue more susceptible to activation by certain proinflammatory
damage. IL-16 is such a chemoattractant molecule. It binds cytokines such as leukoregulin, IL-1, and CD154. Thus, in the
exclusively to CD4 and therefore only influences the migra- context of orbital tissues being infiltrated with lymphocytes
tion of cells displaying CD4 on their surface (326). IL-16 is and other bone marrow-derived cells, the fibroblasts in res-
synthesized as a 69-kDa precursor molecule and is secreted idence appear to respond more robustly to cytokines and
as a 56-kDa protein composed of four identical subunits. The other factors produced by the immune competent cells than
release of IL-16 protein from CD8 T cells and fibroblasts is are other types of fibroblasts. Unfortunately, the vast ma-
dependent on the activity of caspase-3, a cysteine protease jority of evidence supporting this hypothesis has been gen-
(327). At the cell target, IL-16 induces the expression of IL-2 erated from cells in culture. Although these findings tend to
receptors and influences IL-2-dependent cell activation. depict orbital fibroblasts as exhibiting certain behavior, con-
IL-16 has been implicated in a number of human autoim- clusive proof awaits results of studies conducted in vivo in
mune diseases, including rheumatoid arthritis (328), inflam- either human beings or animal models.
E. Fibroblasts from the orbit synthesize high levels IL-1 and leukoregulin up-regulate all three transcripts sev-
of hyaluronan eralfold above basal abundance. The effect of IL-1 was tran-
sient, with the peak increase in mRNAs occurring 6 12 h
A hallmark feature of the connective tissue remodeling
after addition of the cytokine into the culture medium. More-
encountered in GO and dermopathy is the accumulation of over, the state of cell confluence appeared to influence the
the linear polymer, hyaluronan (262). The enormous water magnitude of response to IL-1. A substantial induction of
binding capacity of hyaluronan attracts hydration of con- HAS2 also occurred after treatment with epidermal growth
nective tissue and accounts at least in part for the expansion factor-1 (216). Glucocorticoids, which attenuate cytokine-
of tissues and the expulsion of the eye beyond the normal dependent hyaluronan synthesis in orbital fibroblasts, can
boundaries of the bony orbit. The fibroblast is an important block the induction of HAS by IL-1 and leukoregulin. It is of
source of hyaluronan and the other abundant GAGs such as potential importance that human fibroblasts express sub-
heparin, chondroitin sulfate, and dermatan sulfate (262). Al- stantial levels of all three transcripts. The substrate require-
though these cells produce substantial amounts of sulfated ments for each enzyme appear to be similar, although the
GAG under most culture conditions, the role of these com- profile of hyaluronan chain lengths from each isoform may
plex sugars in the pathogenesis of GO is uncertain. Moreover, differ. Why the same cells should express multiple similar
the most important impact of cytokine action on orbital fi- enzymes is uncertain but could relate to their potentially
broblast GAG synthesis relates to changes in hyaluronan differing roles in development or response to molecular sig-
production levels (214, 311). Unfortunately, no stringent and nals. The enzyme immediately upstream from the HAS pro-
quantitative analysis of the GAG content in the orbital con- teins, UDP-GD, is critical to the synthesis of hyaluronan,
nective tissues affected by GO currently exists. Most evi- chondroitin sulfate, and heparan sulfate. It has recently been
dence for disruption of the normal profile of complex car- cloned and found to be inducible by cytokines in orbital
bohydrates in GO rests on rather old reports utilizing fibroblasts (274). That induction is also time-dependent.
nonspecific and highly subjective methodologies. Thus, Moreover, cycloheximide could induce the transcript after
studies involving assessment of biosynthetic profiles found 6 h of treatment, suggesting that the UDP-GD acts as an
in cultured orbital fibroblasts provide the best clues con- immediate early gene in orbital fibroblasts. Whether this
cerning the net contribution of this cell type to the changes enzyme might ultimately prove rate-limiting under at least
seen in tissue economy relevant to GO. Extrapolations made some tissue conditions is not currently known. Moreover, the
to in vivo disease processes might be misleading. Clearly, a cell signaling upstream from either HAS or UDP-GD in fi-
careful analysis of GAG content in involved orbital tissues broblasts has not been identified. These cascades might
would provide important insights. Moreover, the contribu- prove important targets for the therapeutic intervention in
tion to net GAG synthesis of other cell types residing in the GO and other conditions in which hyaluronan accumulation
orbit, such as the cells of the extraocular muscles and the becomes disordered.
endothelium, need be assessed. In addition, the potential Another important factor governing the accumulation of
roles for these and other cells, such as those derived from the ECM components in GO relates to the degradation of GAGs.
bone narrow, in degrading GAG needs to be defined because Orbital fibroblasts do not express hyaluronidase in culture
alterations in disposal could account for the accumulation (277), a characteristic of other human fibroblasts (216, 269,
seen in GO. 276). When fibroblast monolayers are incubated with me-
When orbital fibroblasts are treated with proinflammatory dium containing radiolabeled hyaluronan, the molecule re-
cytokines, they synthesize high levels of hyaluronan. The mains intact for many days with no evidence of degradation,
levels of production are considerably higher than those regardless of the treatment. Whether these cells express hya-
found in dermal fibroblasts. When orbital fibroblasts are luronidases in situ but lose their ability to degrade the GAGs
exposed to IFN-, the increase in hyaluronan accumulation in culture is uncertain. Alternatively, other cells, either res-
is approximately 50%, whereas the effect is absent in dermal idential or those trafficked to the orbit during inflammation
cultures (311). Leukoregulin on the other hand increases or fibrosis, are involved in hyaluronan turnover. The net
hyaluronan synthesis by up to 15-fold (214). The induction increase in hyaluronan associated with the pathogenesis of
is dependent on de novo protein synthesis and can be atten- GO is clearly of considerable importance in disrupting the
uated with dexamethasone. Moreover, hyaluronan produc- spatial relationships ordinarily maintained between orbital
tion is not influenced when cells are treated with inhibitors contents.
of PG synthesis (214). Pulse-chase studies reveal that hya-
luronan decay is nil in these cultures, indicating that net
F. Orbital fibroblasts display high levels of CD40, an
synthesis is increased under cytokine-induced conditions.
important activation molecule
The mechanisms involved in hyaluronan synthesis have
been elucidated recently with the identification and cloning CD40 is a cell-surface glycoprotein receptor first found
of three members of the HAS family, designated HAS1, 2, displayed on B cells (335). It is a member of the TNF-
and 3 (271273). These proteins are thought to catalyze the receptor superfamily and has subsequently been identified
same rate-limiting and terminal reactions leading to the ini- on other cell types including dendritic, epithelial, and mast
tiation and chain elongation of mature hyaluronan mole- cells, and T lymphocytes. CD40 has an important role when
cules. In orbital fibroblasts, the most abundant HAS mRNA displayed on B cells in that lymphocyte activation is initiated
is that encoding HAS2 (217). Although the levels of the re- through its ligation with CD154, also known as CD40 ligand
spective mRNAs are quite low in unprovoked fibroblasts, (336). CD154 is expressed on T cells, mast cells, and some
tumor cells. The CD40/CD154 activation bridge is thought to A recent report from Pritchard et al. (342) indicates that
represent a critically important mechanism through which T sera and IgGs from patients with GD can provoke the ex-
lymphocytes are believed to activate B cells. More recently, pression of T cell chemoattractant activity. When the fibro-
fibroblasts from synovium, lung, and orbital connective tis- blasts are exposed to GD-IgG and the resulting conditioned
sue have been shown to express high levels of CD40. When medium is then used to treat target T cells, substantial lym-
CD40 is ligated with CD154, fibroblasts from the lung (337) phocyte migration occurs. The vast majority of this activity
and orbit (338) express high levels of PGHS-2 and generate can be attributed to IL-16 and RANTES. GD-IgG activate the
extraordinary amounts of PGE2. The induction of PGHS-2 by fluoride-resistant acid phosphatase/mammalian target of
CD154 is susceptible to glucocorticoids, and these steroids rapamycin/p70s6 k pathway in fibroblasts from patients with
completely abolish the increase in gene expression. More- GD (342). In so doing, the expression of IL-16 is dramatically
over, this induction is mediated through the MAPK pathway increased. IL-16 mRNA is abundant in untreated human
in orbital fibroblasts (338). It would appear that the inter- fibroblasts, but the transcript is not translated until the cells
mediate induction of IL-1 is critical to the up-regulation of are activated (334). The steady-state levels of IL-16 mRNA
PGHS-2. Although IL-1 is induced in a time-dependent fail to increase after GD-IgG activation and may decrease
manner, IL-1 is not expressed after CD40 engagement. This several hours later. Specific inhibitors of the FRAP/mTOR/
selective utilization of IL-1 gene usage in the CD40-activated p70s6 k pathway such as rapamycin block the induction of
orbital fibroblast is of substantial mechanistic importance in IL-16 in the fibroblasts (342). In contrast, the induction
that IL-1 can be induced when these cells are treated with of RANTES appears to be mediated through the activation of
IL-1 or leukoregulin. A number of other genes of potential another pathway and results in the up-regulation of its
relevance to GO are also up-regulated as a consequence of mRNA. Rapamycin fails to block the induction of RANTES.
CD40 ligation in orbital fibroblasts. These include IL-6 and Importantly fibroblasts from several anatomic areas of pa-
IL-8 (339). IL-6 has been implicated in the pathogenesis of the tients with GD are activated to express T cell chemotaxis
thyroid glandular components of GD. IL-8 is a C-X-C che- activity, including areas not usually manifesting clinical dis-
mokine that is expressed and released by hematopoietic and ease. Thus, these results suggest that fibroblast activation in
nonhematopoietic cells at sites of tissue injury and chronic GD may be global. Implicit in the findings to date is the
inflammation. IL-8 also possesses powerful neutrophil and possibility that lymphocytes are trafficked to most if not all
lymphocyte chemoattraction activities. Thus, the selective tissues in GD and that other aspects of the fibroblast phe-
expression and display of CD40 by fibroblasts from certain notype vary between different tissues. Although IL-16 and
tissues and anatomic regions suggests that these cells might RANTES have yet to be directly implicated in GD, the finding
respond differently to signals emanating from T cells, and that both can be selectively induced by disease-specific IgG
this could represent the basis for anatomic site-selective dis- suggests that such a mechanism might play some role in T
ease manifestations. cell infiltration in vivo. Those factors relating to the tissue-
specific targeting of the orbit may relate to the finding that
proinflammatory cytokines and the activated CD40/CD154
G. Do GD-specific IgGs activate fibroblasts?
bridge elicit responses that differ in magnitude and quali-
Fibroblasts from patients with GD have been examined tatively in orbital and nonorbital fibroblasts.
extensively in culture for their potential to respond to specific
IgGs generated in the disease. An early study of Rotella et al.
(340) revealed that IgGs from a number of patients with GO XIV. Future Perspective
could enhance collagen synthesis in normal human fibro-
blasts derived from the skin of the arm. The increase was seen A number of treatments currently are available for the
with some but not all monoclonal antibodies generated from effective treatment of hyperthyroidism associated with GD.
human/mouse hybridomas. More recently, Heufelder and However, in most patients, the thyroid gland is eventually
Bahn (341) reported that IgGs from patients with GO could destroyed due to radioiodine ablation, surgery, or as a con-
increase intercellular adhesion molecule-1 (ICAM-1) expres- sequence of the underlying inflammatory processes. Thus,
sion in orbital fibroblasts from patients with GD but not in patients are subjected to lifelong thyroid hormone replace-
cultures derived from donors without known thyroid dis- ment. A more desirable therapy would be to treat the disease,
ease. That report suggested that certain cytokines could also leaving a functional thyroid intact. This appears feasible in
induce ICAM-1 expression in orbital fibroblasts and that the GD because the initial perturbation is primarily due to TSH
induction of this molecule was accompanied by important agonist activity of stimulatory antibodies with no apparent
functional events related to cell adhesion. The study focused glandular destruction. This is unlike other well-characterized
on the IgG and sera from patients with GO. The question of autoimmune diseases such as multiple sclerosis (experimen-
whether patients with GD but without orbital manifestations tal autoimmune encephalomyelitisa mouse model for mul-
could also induce ICAM-1 expression was left unexplored. tiple sclerosis), Hashimotos thyroiditis, and type-1 diabetes,
Also absent was an assessment of whether nonorbital fibro- where either severe target organ damage precedes the clin-
blasts from patients with GD could respond to GD-derived ical onset of these diseases or the target organs have very
IgG. Thus, it is possible that GD-specific IgGs might activate little or no regenerative capacity. Although these diseases
fibroblasts from patients with the disease, regardless of can be treated to either slow or stop further progression, they
whether they derived from anatomic regions manifesting the might be more difficult to cure. In contrast, if GD can be
disease. diagnosed early, then it might be possible to reverse the
disease using a number of different immune therapy ap- References

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00/0 Endocrine Reviews 24(6):802 835

Current Perspective on the Pathogenesis of Graves

I. Introduction B. Recent studies on the pathogenesis of experimental

G RAVES DISEASE (GD) is a well-characterized disease

FIG. 1. Antigen-processing pathway

FIG. 2. Patient with severe GO. Proptosis,

FIG. 3. Histological examination of or-

FIG. 4. Lower extremities of a patient with

FIG. 5. Schematic diagram of a proposed

FIG. 6. The biosynthesis of the GAG, hyaluronan

disease using a number of different immune therapy ap- References

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