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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
Correspondence: Address all correspondence and requests for reprints to: Bellur S. Prabhakar, Ph.D., Professor and Head, Department of Microbiology and Immunology (MC790), College of Medicine, University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, Illinois 60612. E-mail: Bprabhak{at}uic.edu
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Abstract |
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 Top Abstract I. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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Pathological findings in the thyroid reflect the hyperthyroidism associated with GD. A salient feature of thyroid pathology is hypertrophy and hyperplasia of the parenchyma. A change in the thyroid epithelium is a common finding, with the appearance changing from cuboidal to columnar with papillary infoldings. In addition, these cells demonstrate enlarged Golgi, increased numbers of mitochondria, and vacuolation of the colloid. These changes in the thyroid are often accompanied by a diffuse cellular infiltration consisting of lymphocytes and plasma cells. Occasionally, the infiltration can be more focal and resemble changes seen in Hashimoto’s thyroiditis.
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II. Risk Factors for Developing Graves’ Disease (GD) |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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A. Genetic factors
Increased incidence of GD among members of a family indicates that genetic factors might play an important role in determining susceptibility to GD (1, 2). Studies involving twins indicate a higher degree of concordance, suggesting that the genetic factors might be a major contributor (3). Earlier studies have shown that patients with GD express human leukocyte antigen (HLA)-B8 more often than the control subjects without the disease (4). Other studies have shown that the risk of developing the disease is higher 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 HLA DR ß1*07 appears to confer protection (9). However, there is racial variation in the association of MHC haplotypes with increased risk for the development of GD (9). Two recent reviews on genetic susceptibility to GD have summarized a large number of studies to date (10, 11). The general conclusions one can draw from studies to date are that multiple genetic factors appear to contribute to the risk of developing GD and the penetrance of the disease is about 30% in monozygotic twins. Some of the susceptibility genes are involved in the generation of immune responses and/or associated with X chromosome, whereas others appear to be specific to GD (e.g., GD-1, -2, and -3).
How could these gene products play a role in protecting against or enhancing the risk for the development of the disease? Hyperthyroidism in GD is mediated by autoantibodies to TSHR protein, and the protein antigens are T cell-dependent antigens. Therefore, CD4+ T cells, most likely, play an important role because they provide the necessary help for autoantibody production. If this were true, then the most effective antigen presentation would be in the context of MHC class-II molecules, and thus they may play a critical role in the pathogenesis of GD. MHC class-II molecules form peptide binding grooves, the specificity of which is determined by their amino acid sequence. This allows binding of antigenic peptides of a certain length and with specific sequences. T cells, bearing cognate receptors, in turn recognize these MHC-peptide complexes and undergo activation. Thus, MHC molecules play a very critical role in the antigen presentation and thus in the T cell repertoire generation.
Early in the thymocyte development, immature T cells migrate from bone marrow into the thymus where they undergo 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 because many tissue-specific antigens may not be expressed on the thymic epithelium. However, cells capable of interacting with exogenous antigens and some cells that can interact with self-antigens are positively selected and migrate to the periphery (12). Because a given MHC molecule can bind only a limited number of peptides with certain sequences, they largely determine the repertoire of T cells that are either eliminated in the thymus (negative selection) or allowed to migrate to the periphery (positive selection). Because individuals expressing HLA DR3 and DQA1*0501 haplotypes have an increased risk of developing the disease, it is possible that they might fail to present certain tissue-specific pathogenic peptides in their peptide binding groove and thus prevent negative selection of self-reactive T cells that express the cognate T cell receptor (TCR) on their surface. These self-reactive T cells could encounter the antigen in the periphery and undergo activation. In contrast, HLA DR ß1*07, which confers protection, might bind pathogenic peptides and facilitate negative selection of self-reactive T cells, expressing cognate TCRs, in the thymus and prevent an autoimmune response. Thus, MHC gene products can differentially affect both the repertoire selection in the thymus and subsequent autoimmune responses in the periphery.
More recently, a number of reports have suggested an association between cytotoxic T lymphocyte antigen-4 (CTLA4) and GD. The CTLA4 protein exists in multiple isoforms due to genetic polymorphism in the first exon (13). This is often linked with another polymorphism of AT dimers at the 3'-untranslated region of the third exon, which is thought to be associated with increased risk of GD (14, 15). Similarly, other associations with genetic polymorphism in CTLA4 have been reported (16, 17, 18). How could this molecule be linked with increased susceptibility to GD? T Lymphocytes constitutively express a costimulatory molecule called CD28 on their surface. During the initiation of an immune response, the T cells receive their first signal through their TCR interaction with the MHC-peptide complexes on the antigen-presenting cells (APCs). However, naive lymphocytes require a second signal to undergo activation that is delivered through CD28 upon its interaction with specific ligands, B7.1 and B7.2 (i.e., CD80 and CD86, respectively), expressed on the surface of APCs. Subsequent to activation, T cells up-regulate CTLA4 expression on their surface. The CTLA4, which is structurally related to CD28, not only interacts with CD80 and CD86, but does so with a 100-fold higher affinity. Because of higher affinity, CTLA4 competes with CD28 for binding to CD80 and CD86 and causes down-modulation of T cell responses due to negative signaling and/or lack of continued positive signaling because of competition with CD28 for ligand binding. A defective interaction of CTLA4 with its ligands could result in a generalized defect in the down-modulation of immune responses. Therefore, although the observations on CTLA4 polymorphism are of considerable interest, at this time it is not clear how a defect in this gene could specifically enhance immune response to TSHR and susceptibility to GD.
B. Environmental factors
Factors such as genetic background, age, and gender partially account for the development of GD. As with other autoimmune diseases, an environmental factor has long been suspected in the etiology of GD. If environmental factors such as bacteria are involved, how would such an agent initiate an autoimmune response to a specific self-antigen? Cells other than APCs do not express MHC class-II and thus are not capable of presenting antigens to CD4+ T cells. Similarly, most cells show either little or no expression of costimulatory molecules and thus fail to provide the second signal to antigen-specific T cells, which results in either a failure to activate T cells or induction of anergy. There are a number of potential mechanisms by which an environmental agent could trigger an autoimmune response. These include induction of an inflammatory response leading to the production of proinflammatory cytokines that can cause enhanced/aberrant expression of MHC class-II and costimulatory molecules. This could lead to an otherwise innocuous presentation of self-peptide by MHC molecules into an effective antigen presentation and cause activation of antigen-specific T cells. Thus, cytokine production and/or an imbalance in cytokines caused by infection could lead to the initiation of an autoimmune response. The role of cytokines in GD has been discussed in a recent review (19), and the role of cytokines as it relates to GO will be discussed later. Microbial infections can also cause overexpression (e.g., heat shock proteins) and/or altered expression of certain self-proteins (altered self), which will either provide the necessary strength of signal or be perceived as foreign (20).
Many microbial products can act as either B cell or T cell mitogens and cause nonspecific activation of lymphocytes, including self-reactive lymphocytes. Once self-reactive lymphocytes are activated, they can continue to persist and expand through antigenic stimulation from self-antigens. This is because the strength of antigen presentation required to sustain an anti-self response is lower than that required to initiate an anti-self immune response. A number of bacteria-derived proteins have now been identified as superantigens and linked to Kawasaki disease (21), rheumatoid arthritis (22), and other autoimmune diseases (23). Although the exact mechanism of their action is not known, superantigens have been postulated to facilitate development of autoimmune diseases by: 1) activation of a diverse population of T cells with different fine specificities, including self-reactive T cells; and/or 2) polyclonal activation of B cells, including those that produce autoantibodies. Polyclonal activation of B cells resulting in production of antibody has been shown for Mycoplasma arthriditis mitogen (24) and toxic shock syndrome toxin-1 (25). Production of autoantibodies is thought to be mediated by bridging of superantigen to CD4+ T cells and B cells. Superantigens have been identified from culture supernatants from Yersinia enterocolitica (26) and Y. pseudotuberculosis (27), and these infections have been linked to Reiter’s syndrome, reactive arthritis, GD, and ankylosing spondylitis (28). Although we have a much better understanding of superantigens and the mechanisms by which they activate lymphocytes, we know very little about the role of Yersinia or Yersinia-derived superantigens and/or mitogens in different disease states including GD. It is possible that Yersinia-derived T cell superantigens and B cell mitogens might act independently or in combination to activate T cells and/or B cells, resulting in preferential expansion of B cells recognizing cross-reactive epitopes on TSHR and Yersinia. Such B cells will not only produce autoantibodies reactive with TSHR, but could also serve as efficient APCs that could present TSHR peptides to self-reactive T cells and perpetuate anti-TSHR responses.
Molecular mimicry is one of the most commonly invoked and studied mechanisms for the induction of autoimmunity. A finite probability exists for two proteins sharing common epitopes formed either by a primary sequence of identical amino acids or by noncontiguous amino acids adjacent by virtue of three-dimensional conformation, and resulting in molecular mimicry. After infection, the host mounts a brisk response against the microbe, and this response might become directed against a self-antigen that can be sustained even after the clearance of the microbial agent due to continued antigenic stimulation by the endogenous host protein. This response then can expand due to epitope spreading and eventually cause the disease. Molecular mimicry has been insinuated in other examples of autoimmunity including HLA-B27 and Klebsiella in ankylosing spondylitis (29), myelin basic protein, and mouse hepatitis virus in experimental allergic encephalomyelitis (30), cardiac myosin, and coxsackie virus B4 in myocarditis (31, 32) and others (33). Primary amino acid sequence homology between self-peptides and the foreign antigens as well as conformational similarities have been demonstrated (30, 32, 33).
C. Y. enterocolitica and GD
Due to seasonal trends and geographic variations in the incidence of GD (34, 35), it has been suggested that infectious agents might be involved in triggering the breakdown of tolerance to TSHR in patients with GD. In addition to the reports of increased susceptibility to infections in individuals who fail to secrete certain ABO blood group antigens into the saliva (36, 37), serological evidence for a recent bacterial or viral infection has been reported in a high number of newly diagnosed patients with GD (38).
Y. enterocolitica has been postulated to play a role in the induction of GD via molecular mimicry (39, 40, 41). This speculation is supported by studies reporting the presence of antibodies against Y. enterocolitica in a high proportion of patients (i.e., 72–81%) with autoimmune thyroid disease (42, 43, 44, 45). In addition, Y. enterocolitica has been shown to have binding sites for TSH (46), and antibodies isolated from GD patients could inhibit TSH binding to Yersinia (47) and react with a 64-kDa protein, which was thought to be the bacterial protein to which TSH binds (47). Moreover, antibodies raised against thyroid membranes have been shown to bind Y. enterocolitica (48), and outer membrane proteins (YOPs) encoded by a virulence plasmid of Yersinia have also been linked to the induction of GD (49). Wenzel et al. (50) reported that 72% of patients with GD and 81% of patients with recurrent GD had antibodies to YOPs, whereas only 35% of unmatched controls had such antibodies, and YOPs could induce antibodies in rabbits that recognized components of thyroid epithelial cells. However, data reported by Arscott et al. (51) suggested that there was no unique serological reactivity against YOPs or other Yersinia membrane antigens and that any causal relationship between Yersinia infection and GD may be related to activation of cross-reactive T cells leading to breakdown of self-tolerance to TSHR. Furthermore, Ebner et al. (52) reported that rats immunized with Y. enterocolitica-purified YOPs developed thyroiditis. In this regard, T cell-mediated immunity toward Y. enterocolitica has also been reported in patients with GD (53). Burman et al. (54) reported that TSHR-specific cDNA probe could hybridize to DNA from Y. enterocolitica under low stringency conditions, indicating that they may share stretches of common sequences. However, to date, a causal relationship between Yersinia infection and the development of GD has not been fully established (41, 42).
D. Cross-reactivity of Y. enterocolitica proteins with TSHR
Several years ago, Seetharamaiah et al. (55, 56) expressed the extracellular domain of human TSHR (ETSHR) using a baculovirus expression system. ETSHR protein was purified to homogeneity using HPLC, and upon refolding it could bind TSH (55) and anti-TSHR antibodies in patient sera (56). Mice immunized with ETSHR developed some clinical features characteristic of GD, including high levels of antibodies to the TSHR, with a concomitant elevation in T4 levels (57).
Using this recombinant ETSHR, cross-reactivity of Y. enterocolitica with ETSHR was explored. Initial studies showed that antibodies produced in rabbits and mice to purified ETSHR reacted with envelope preparations from Y. enterocolitica. This cross-reactivity was specifically blocked by purified ETSHR or an envelope preparation of Y. enterocolitica. Moreover, antibodies reactive with ETSHR were readily induced in mice by immunizing with Y. enterocolitica, but not with other Gram-negative or Gram-positive bacteria. These studies provided the first direct evidence that immunization with Y. enterocolitica can lead to the production of antibodies capable of reacting with TSHR (39).
Subsequently, the envelope protein(s) responsible for cross-reactivity to ETSHR was identified using anti-Yersinia antibodies that specifically bound to the ETSHR protein (50-kDa band) on a Western blot. The two low-molecular weight Yersinia envelope proteins (5.5 kDa and 8 kDa) that cross-reacted with the ETSHR, TSHR-cross-reactive protein (TSHR-CRP), were gel purified (40) and used for immunizing CBA/J, C57BL/6 and BALB/c mice. Sera obtained from these mice recognized both ETSHR protein and TSHR-CRP in an ELISA and Western blot (58). Subsequently, it was shown that TSHR-CRP was mitogenic for spleen cells (58) from C3H/HeJ mice, which are lipopolysaccharide nonresponders, and was pronase-sensitive (58). Cell proliferation studies using B cell- and T cell-enriched populations showed that these protein(s) are mitogenic to B cells, and not to T cells, and could induce production of high levels of IL-6 and significant amounts of IgG and IgM by B cells. These results indicate that TSHR-CRPs, which contain epitopes that cross-react with TSHR, represent highly effective B cell mitogens, and the mitogenic activity was not due to lipopolysaccharide contamination. Alignment of peptide sequences showed that TSHR-CRP is homologous to bacterial lipoproteins (LP). Although, all Gram-negative bacteria produce LP, cross-reactivity of Y. enterocolitica LP with TSHR was unique (59). Moreover, LP was capable of inducing high levels of IL-6 and IL-8 production by human B cells. These observations have significant implications for understanding the role of molecular mimicry in the breakdown of self-tolerance and generating autoimmunity to TSHR (59).
In a recent review, Benoist and Mathis (60) raise some important issues to consider with regard to the role of microbial agents in promoting T cell-mediated autoimmunity. Although a number of animal models have provided convincing evidence that a given autoimmune disease can be induced by a particular microbe, they argue that many of the associations are less convincing based upon five different criteria they propose for "air-tight" cases (which are modifications of previously presented concepts). Although they raise some important points with regard to the role of molecular mimicry for us to consider, their perspective is restricted to issues involving T cell epitope recognition and diseases in which T cells, and not B cells, act as effector cells. Moreover, it should be noted that molecular mimicry is only one of several potential mechanisms that a microbe can use to induce autoimmunity. As discussed in Section II.B, microbial agents can cause immunological perturbations through their mitogenic activity, ability to alter self-proteins and/or induce proinflammatory cytokines, and direct up-regulation of MHC gene products and costimulatory molecules (bystander effects). Needless to say, as in all other diseases, including metabolic disorders, cancer, etc., it is not easy to directly demonstrate the role of environmental factors in autoimmunity. As argued by Benoist and Mathis (60), more experimental evidence is needed to fully establish the role of a given microbial agent in the initiation of autoimmunity and autoimmune diseases. With the availability of an animal model, now it might be feasible to investigate the potential immunomodulatory effects of Y. enterocolitica and/or the consequence of molecular mimicry that it exhibits with TSHR on the development of GD.
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III. Immunological Basis for GD |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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Although, imbalances in T cell ratios have been proposed for the regulation of anti-TSHR antibody production (79, 80), other studies have reported contradicting results (81, 82, 83). Because of the unavailability of purified TSHR, the antigen specificity of T cells could not be determined, and thus it is difficult to assess the significance of changes in the total T cell ratios. More recent studies have analyzed the TCR gene utilization in lymphocytes infiltrating the thyroid gland (84, 85). The rationale for these studies was that cells infiltrating the thyroid gland are likely to be thyroid antigen-specific and that utilization of a restricted number of TCR genes by these infiltrating T cells would suggest that a limited number of T cell epitopes are involved. Data from these studies showed that early after onset of GD there is preferential usage of certain TCR variable (V)
genes (84). However, this study and others also showed that there was considerable variation in TCR usage among different patients (84, 86). This is not surprising, considering the fact that T cells recognize antigens in the context of MHC molecules. Because different patients might have different HLAs, they need different TCRs to recognize the antigenic peptide (87). The HLA variation, coupled with the lack of information on antigen specificity of infiltrating cells, makes the interpretation of these data difficult. A putative role for T cells in GO will be discussed later.
Passive transfer of Igs from GD patients to experimental animals causes increased thyroid hormone production (88). 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 protein (91), it has been very difficult to purify the TSHR from thyroid membranes. Thus, to date, it has not been feasible to fully evaluate the importance of the idiotypic network for the regulation of TSHR-specific antibody production. Based on studies published thus far, there exists little or no evidence to suggest that an idiotypic network plays a critical role in the regulation of immune responses to TSHR.
Recently, considerable efforts have been made to understand the role of apoptosis in both Hashimoto’s thyroiditis and GD. It appears that thyroid-infiltrating lymphocytes in patients with Hashimito’s thyroiditis express little or no Fas but exhibit high levels of Bcl-2, whereas their thyrocytes show decreased Bcl-2 and increased Fas as well as Fas ligand. This would render thyrocytes susceptible to Fas-mediated apoptosis and facilitate thyroid destruction. However, in contrast, patients with GD show increased Bcl-2 and decreased Fas expression on their thyrocytes, while exhibiting increased Fas and decreased Bcl-2 on their lymphocytes. This would lead to apoptotic death of infiltrating lymphocytes and sparing of thyroid from the effects of inflammatory responses. Recent papers (92, 93, 94) on this topic have discussed the role of apoptosis in thyroid autoimmunity; therefore, we will refrain from a detailed discussion here.
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IV. Autoimmune Responses to Thyroid Autoantigens |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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In 1989, a cDNA-encoding dog TSHR was cloned (95) followed by cloning of human and rat cDNAs (96, 97, 98). This resulted in numerous studies aimed at understanding the structure-function relationship of the protein aimed at the determination of various binding sites for TSH as well as functionally different antibodies (99, 100, 101, 102, 103). Many initial studies used synthetic peptides that were derived from the protein sequence predicted from the cDNA sequence and provided some insights into the potential antibody binding epitopes (99, 100, 101, 102, 103). Initial efforts to express the protein in bacteria resulted in limited yields of protein that was not folded properly. Subsequent studies using an insect cell expression system resulted in higher yields of both glycosylated and nonglycosylated proteins representing the ETSHR. Studies utilizing these protein preparations clearly showed that the ETSHR was sufficient for the TSH and patient autoantibody binding, and that glycosylation was required for autoantibody recognition (104, 105). Although a small fraction of the protein produced in insect cells acquired appropriate conformation, as indicated by TSH and autoantibody binding, this expression system was unsuitable for the production of TSHR with native conformation because the protein remained largely aggregated. Therefore, many studies, aimed at determining the functionally relevant epitopes on TSHR were conducted using two major approaches involving transfection of mammalian cells with various cDNA constructs. One involved construction of TSHR/LH/choriogonadotropin receptor chimeras in which select segments of TSHR were replaced with the corresponding segments from the LH/choriogonadotropin receptor. Studies from this approach resulted in the identification of regions of TSHR that are critical for TSH and blocking and stimulatory antibody binding. Another approach has been to use deletion mutants and/or site-directed mutagenesis to identify the functionally relevant regions of TSHR. These studies revealed that certain glycosylation sites and cysteine residues are absolutely essential for proper folding of the receptor. Because of limitations associated with each of the approaches mentioned above, no single approach has yielded conclusive results on the overall structure-function relationship of the TSHR. However, collectively, these studies have led to several important conclusions. Both TSH and autoantibodies can primarily bind to the ETSHR, and the TSH binding epitope consists of discontinuous amino acids that come together due to protein folding. Antibodies against one or more epitopes can inhibit TSH binding or TSH-mediated activation of cells by affecting a step subsequent to TSH binding. The stimulatory autoantibody binding sites reside predominantly at the N terminus of the protein, whereas the blocking antibodies bind primarily to the C terminus. Glycosylation of the receptor is important for proper folding of the receptor and autoantibody binding. However, it is not clear whether autoantibodies directly interact with the sugar moieties or with epitopes that are formed as a result of proper glycosylation of the receptor protein. A number of excellent reviews (99, 100, 101, 102, 103, 106) have dealt with the structure-function relationship of TSHR; therefore, we will not discuss this issue any further in this paper.
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V. TSH Receptor as an Autoantigen |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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In addition to the subunit structure of TSHR, other important immunological factors must be required to induce an autoimmune response. Although self-reactive T cells might escape negative selection in the thymus, they can be kept in an inactive state through a number of different peripheral tolerance mechanisms (12). For an effective antigen presentation, not only does the antigenic peptide have to be presented by the MHC molecule, but the strength of signal is also very important. This is referred to as the first checkpoint, is demonstrated in animal models of diabetes, and is a consequence of suboptimal presentation of antigens (110, 111). Even if an appropriate signal is delivered, it has to be accompanied by a second signal to activate naive T cells. It is generally accepted that self-peptides do not efficiently activate APCs (i.e., lack of costimulation) and thus can induce T cell anergy rather than activation (111, 112). An even more important mechanism is the engagement of CTLA4, which down-modulates the immune response (113, 114, 115, 116, 117, 118). More recently, another molecule called PD1 (programmed cell death) has been shown to suppress cytokine production through cell cycle arrest and induce T cell anergy (119, 120, 121, 122, 123, 124). Yet another mechanism that can help maintain tolerance in the face of appropriate antigen presentation is phenotypic skewing of helper T cells (125, 126, 127, 128). There are two types of helper T cells, namely Th1 and Th2. Cytokines produced by Th2 cells play a more important role in antibody production than the cytokines produced by Th1 cells. Therefore, if Th1 cells instead of Th2 cells are activated, it is unlikely that it will result in a pathogenic antibody response against the TSHR. If appropriate self-reactive T cells are activated, still they can be deleted by activation-induced cell death (129, 130, 131, 132, 133). Self-antigens are not readily cleared, and therefore they can provide repetitive and continuous stimulation to T cells, causing them to undergo activation-induced cell death through Fas ligation by Fas ligand. Therefore, yet unknown perturbations in the immunoregulatory mechanisms of the host might contribute to the pathogenesis of GD.
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VI. Evolution of Autoimmune Response to TSHR |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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TSHR is a member of the large family of guanine-nucleotide binding (G) protein-coupled receptors. The full-length TSHR cDNA consists of an open reading frame encoding 764 amino acids, including a 21-amino acid signal peptide, a large extracellular domain, seven transmembrane domains, and a short cytoplasmic tail (95, 96, 97, 98). The ETSHR forms the binding site for TSH on the outside surface of the thyroid cell membrane, and the cytoplasmic domain interacts with the regulatory subunits of adenylate cyclase. Initially, high levels of the nonglycosylated ETSHR were produced using a baculovirus expression system (55). This protein was purified to homogeneity using reversed phase HPLC. The purified ETSHR protein was refolded and shown to specifically bind 125I TSH in a dose-dependent manner (56).
A. Early studies on pathogenesis of experimental GD
The insect cell-derived protein was used to successfully produce antibodies capable of blocking TSH binding to native TSHR (55). These studies suggested that the ectodomain was sufficient for TSH binding, albeit at a lower affinity, and for the induction of TSHR-specific antibodies. To identify a strain of mice susceptible to the development of hyperthyroxinemia, C57Bl/6J, BALB/CJ, B10BR/SgSnJ, and SJL/J mice were immunized with ETSHR (57). Sera obtained after five immunizations from different strains of mice were TBII-positive, albeit at different levels of significance (57), whereas only sera collected on d 55 from BALB/CJ mice showed significant elevation in T4 hormone compared with sera obtained from the corresponding control mice. However, the T4 elevation was transient and indicated that immunization with ETSHR had failed to break tolerance to self-TSHR and that immunization using TSHR with native conformation may be required.
To address this, BALB/CJ mice were primed with the ETSHR and then challenged with solubilized thyroid membrane. Relative to controls, immunized mice showed high levels of TBII activity and significantly elevated T4 (57). The [131I] uptake by thyroid glands of test mice was found to be higher than in control mice, suggesting that the elevations in free T4 were not a consequence of gland destruction. Histopathological examination of thyroid glands from hyperthyroid mice showed morphological alterations characteristic of hyperactive glands, including hydropic and subnuclear vacuolar changes, as well as focal scalloping of the colloid within isolated acini. No inflammatory activity, interstitial fibrosis, acinar atrophy, or glandular destruction was found, and this further suggested that the elevated T4 levels were due to hypersecretion. These results showed that ETSHR can prime the animal and allow development of antibodies that can cause hyperthyroidism. The limitation of this study was that the mice became euthyroid within weeks after cessation of TSHR injection, again indicating failure to break tolerance to self-TSHR.
In an attempt to develop an animal model for GD, Marion et al. (134) used affinity-purified human TSHR from a human thyroid cell clone designated GEJ to immunize B10S (H-2s), B10 (H-2b), B10G (H-2q), B10 D2 (H-2d), and B10BR (H-2k) male and female mice. The male and female H-2s, male H-2b, and female H-2q mice exhibited lymphocytic infiltration in their thyroid glands. Immunized mice showed transient marginal elevation in T3. Because no data on serum TBII, TSBAb, and thyroid-stimulating antibody (TSAb) activities were provided, it was not possible to conclude whether these mice developed hypothyroidism or hyperthyroidism.
Other investigators have attempted to develop an animal model for autoimmune GD (hyperthyroidism) and thyroiditis (hypothyroidism) using recombinant TSHR. Costagliola et al. (135) used a fusion protein consisting of ETSHR and maltose binding protein (MBP-ECD). Sera or IgG from immunized mice did not show any thyroid-stimulating activity (TSAb), but showed significant TBII and TSBAb activity and lower serum T4 levels compared with mice immunized with MBP alone. Histological examination of thyroids revealed extensive vascularization and an atypical lymphoblastoid infiltration. In a subsequent study, they extended these observations to male and female mice (136). In another study, they immunized female NOD (H2g), CBA (H2k), and C57BL (H2b) mice with MBP-ECD (137). No significant serum TBII, TSAb, and TSBAb were detected in any of the strains of mice. Although there was no lymphocytic infiltration of the thyroid glands of CBA or C57BL mice, all NOD mice had severe thyroiditis. Based on these studies, the authors concluded that H2d mice develop thyroiditis and TBII/TSBAb, H2g mice develop thyroiditis in the absence of functional TSHR antibodies, whereas H2b and H2k mice are resistant. Furthermore, they showed the ability of TSHR-primed splenocytes to transfer thyroiditis to naive recipients (138). However, neither the antibody properties nor the hormonal perturbations were consistent with findings in GD. In many of these studies, although immunization with MBP alone had no significant effect, the influence of the MBP component of MBP-ECD on the induction of thyroiditis was not evaluated. One possible explanation is that because more recent studies have shown that glycosylated ETSHR is required for stimulatory antibody recognition (and presumably for its induction), antibodies raised against the nonglycosylated ECD produced in bacteria might not have been sufficient to cause GD.
Both BALB/c and NOD mice were immunized with MBP-ECD, and unfractionated T cells or CD4+-enriched cells obtained from these mice were stimulated in vitro with the MBP-ECD protein and then adoptively transferred to the corresponding strain of naive recipients. The recipients showed TBII activity in their sera that persisted for 12 wk. NOD mice showed marginal hypothyroidism, whereas most BALB/c mice were euthyroid with few showing some elevation in T4 levels (138). Approximately two thirds of BALB/c mice showed accumulation of adipose tissue and infiltration of mast cells in the orbital tissue. Collectively, these studies showed that multiple factors determine the outcome of immunization with TSHR, and suggested the polygenic nature of the disease (139).
It is apparent that an effective antibody response (i.e., IgG) against the TSHR protein would require help from T cells that have the same antigen specificity (140, 141, 142). This phenomenon is called linked recognition and would require antigen presentation by professional APCs and activation of antigen-specific T cells before autoantibody production by B cells with specificity for the same antigen. However, it is interesting to note that the epitopes recognized by T cells and B cells need not be part of the same protein but have to be physically linked (140, 141, 142). This is important for subsequent antigen presentation by B cells because these cells will internalize the antigen through receptor-mediated endocytosis, process the antigen, and present specific peptides on their surface leading to subsequent antigen-specific T cell activation. This principle has been exploited in the development of an effective vaccine against Haemophilus influenzae type B. The protective antibodies against this bacterial infection are directed against the bacterial polysaccharides. Adults readily produce such protective antibodies, whereas infants are not as efficient. Therefore, the bacterial polysaccharide is linked to the tetanus toxoid against which the infants mount an effective response. In this case, B cells that bind to the polysaccharides can readily produce antibodies with help from T cells specific for peptides from tetanus toxoid because of the linked recognition. In light of this, what is the consequence of using a self-antigen (TSHR) that is fused to a foreign antigen (MBP) to elicit a response against the self-antigen? It is well known that a foreign antigen is likely to be more immunogenic than a self-antigen such as TSHR. If this were the case, it is likely that the immune response will be largely directed against the MBP. In the absence of a clear demonstration of TSHR specificity of the T cells in MBP-TSHR immunized mice, the possibility of MBP-specific T cells providing help to TSHR-specific B cells, through linked recognition, cannot be ruled out.
Carayanniotis et al. (143) attempted to develop an animal model by immunizing C57Bl/10, B10.BR/SgSn, C3H/He, SJL, DBA/I, and BALB/c mice with an ETSHR protein produced in insect cells. All strains of mice showed strong specific T cell proliferative responses. Sera from SJL and BALB/c mice showed good TBII activity. However, there were no significant changes in serum TSH, T4, and iodine uptake. Authors suggested that their inability to induce hyperthyroidism is most likely due to the inappropriate folding of the ETSHR protein. Vlase et al. (144) immunized female BALB/c and CBA mice with human TSHR-ECD produced in Escherichia coli and in insect cells, respectively. Similar to Carayanniotis et al. (143), these investigators failed to induce either hyperthyroidism or thyroiditis. Subsequently, studies from a number of laboratories have shown that insect cells are not capable of removing the human signal sequence from the protein. Because the cDNA used for protein expression in these two studies encodes for a human signal peptide consisting of 21 largely hydrophobic amino acids at the N terminus, it likely affected the conformation of the protein that may be needed for pathogenic antibody production. In another study, Vlase et al. (145) used refolded ectodomain of mouse TSHR produced in insect cells to immunize BALB/c mice. Immunized mice developed considerable TBII and TSBAb activities and exhibited lower levels of T3, but not T4, and higher levels of TSH.
B. Recent studies on the pathogenesis of experimental GD
Shimojo et al. (146) immunized female AKR/N (H-2K) mice with fibroblasts expressing both MHC class-II and a full-length human TSHR. Most of these mice exhibited good serum TBII activity. About 20% of these mice developed hyperthyroidism as evidenced by significantly elevated serum T4 levels. This was similar to earlier findings of severe hyperthyroidism when BALB/c mice were inoculated with ETSHR followed by thyroid membrane preparations (57). Based on these and other results (146, 147, 148), the authors have suggested that expression of MHC class-II on cells that express TSHR can result in the induction of stimulatory anti-TSHR antibodies in a small proportion of animals. The data presented in this study and an earlier study (57) may also suggest that some of the pathogenic epitopes might be present on the native TSHR. A similar study from another group (149) confirmed these observations by showing that approximately 25% of the immunized animals developed hyperthyroidism and one mouse developed hypothyroidism with elevated TSH levels. Mice with hyperthyroidism showed thyroid hypertrophy without lymphocytic infiltration. When mice were immunized with cells expressing TSHR and MHC class-II along with alum as an adjuvant, they showed increased prevalence of the disease (approximately 45%), with a shorter duration required for the onset. However, when TSHR along with complete Freund’s adjuvant was used, approximately 30% of the animals developed the disease with a slower onset. In this study, no apparent difference was found between male and female mice (149).
Additional studies showed that several different strains of mice with different non-MHC background but expressing H-2K haplotype were able to produce TBII antibodies upon immunization with cells expressing class-II and TSHR proteins (148). Unlike the earlier report with AKR mice (147), these studies showed that TBII activity could be induced in CBA and C3H mice using fibroblasts expressing TSHR without the class-II protein. A subsequent study showed that the N-terminal half of the ectodomain might be sufficient to induce TBII activity in AKR mice, but the entire ectodomain was needed to induce TSAb activity in approximately 20% of the animals. To carry out systematic studies on the molecular mechanisms, a higher proportion of animals need to develop the disease, and that may require a more susceptible strain of mice, different antigen preparation, and/or immunization schedule (150, 151).
Results from studies using fibroblasts stably transfected with human TSHR and MHC class-II have been used to argue that aberrant class-II expression is required for disease induction (146). Although this might be correct, there are no conclusive data to support this conclusion. The contention that aberrant expression of class-II is necessary has been based largely on finding that MHC class-II is expressed in both thyroid and islets of affected patients (152). Subsequent studies showed that expression of class-II molecules or exogenous antigens on ß-islet cells using a rat insulin promoter can result in varying outcomes, including development of spontaneous diabetes, and thus lent support for the initial observation (153). This seems reasonable in a T cell-mediated autoimmune disease that results in the destruction of the target tissue because the effector T cells need to recognize the self-peptide/MHC complex on the target tissue. It is unlikely that such an aberrant expression of class-II is required for the induction of an autoantibody response. This proposition is based on the current understanding of T cell and B cell interactions required for an effective antibody production (Fig. 1
). The initial step in an immune response against a T cell-dependent antigen is phagocytosis of the antigen in the periphery by professional APCs, most likely dendritic cells (DCs). Once they pick up the antigen, DCs migrate and begin processing the antigen in the T cell zone of the regional lymph node. Here, antigen-specific T cells, which enter the lymph node through the afferent lymphatics, are trapped by the DCs with relevant peptide-MHC class-II on their surface. Subsequently, as they circulate through the lymph nodes, antigen-specific B cells are trapped where they are activated by antigen-specific T cells to produce antibodies. Antibodies enter the circulation and react with the TSHR, causing hyperthyroidism. In this scenario, it is likely that the availability of appropriately folded protein to B cells might be more important than the expression of class-II molecules on the cells used for immunization. Moreover, a large number of studies have clearly shown that there is little or no priming of naive T cells in the absence of costimulation. Because these RT cells most likely lacked both adhesion and costimulatory molecules (i.e., B7.2), it is not apparent how these cells could have served as efficient APCs to prime naive T cells required for optimal anti-TSHR responses.
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-chain and a ß-chain. Each of the chains consists of two domains. RT cells constitutively do not express class-II molecules and are transfected with a cDNA that can encode a chimeric class-II molecule. The -1 and -2 and ß-2 domains of this chimeric class-II were derived from H-2K haplotype, and the ß-1 domain was derived from H-2D (155). These cells were originally constructed to identify peptide binding sites on class-II and were used in vitro as APCs to test for proliferative responses of T cells primed in vivo by direct antigen inoculation. However, it is not clear whether the chimeric class-II molecule expressed on RT cells could have served as an alloantigen (due to ß-1 domain derived from H-2d) in AKR mice (H-2 k haplotype) leading to activation of alloreactive T cells rather than TSHR-specific T cells, that can provide nonspecific help. This might explain a lack of T cell infiltration in thyroids of mice with severe hyperthyroidism. Nevertheless, these studies are very important because they clearly showed that hyperthyroidism can be induced in experimental animals using a recombinant TSHR. Moreover, they showed that the nature of immune response is directly dependent on the antigen and/or the adjuvant used for inoculation. Thus, immunization using TSHR with native conformation might result in the production of stimulatory antibodies that could then cause hyperthyroidism.
Other studies have reported using cDNA vaccination to induce pathogenic antibodies against the TSHR. These studies yielded somewhat contradictory results depending upon the strain of mice used for immunization. BALB/c mice immunized with the cDNA developed antibodies to TSHR (156). Only one mouse contained TSAb, but all mice, irrespective of their autoantibody status, were euthyroid. Examination of the thyroid glands from these mice revealed nondestructive thyroiditis, with B cell infiltration. When a similar experiment was carried out using NMR outbred mice, all mice developed antibodies to TSHR detected by fluorescence-activated cell sorter (157). Nine of 30 males showed mild hypothyroidism with TSBAb and lower T4, whereas four of 30 females showed signs of hyperthyroidism with elevated T3 and T4 levels and TSAb activity with a concomitant decline in TSH levels. Another study showed a heterogeneous response upon vaccination of wild-type and interferon-
(IFN-) -/- BALB/c mice with TSHR cDNA (158). Further refinement of this approach and the use of female mice might lead to the development of an animal model in which a higher proportion of mice develop GD.
In a more recent study (159), different strains of mice were immunized with adenovirus expressing either TSHR or ß-galactosidase three times at 3-wk intervals, and mice were observed for 8 wk after the third immunization. Fifty-five percent of female and 33% of male BALB/c mice and 25% of female C57BL/6 mice developed hyperthyroidism characterized by the presence of TBII and TSAb activity and elevated T4 levels in the sera. Thyroid glands of affected mice showed hypertrophy with no cellular infiltration, and the extraocular muscles were normal.
Another study reported successful induction of Graves’-like disease in nearly 100% of female BALB/c mice using either mouse or human TSHR proteins (160). BALB/c mice, known to generate Th2-type responses, were used because antibody-mediated autoimmune diseases are currently thought to be driven by Th2 helper T cells. Female BALB/c mice were used because of their expected enhanced susceptibility to GD. The protein was either expressed on the surface of syngeneic or xenogeneic cells or produced in a soluble form. Irrespective of the antigen used for immunization, nearly 100% of the mice developed hyperthyroidism characterized by the presence of high levels of TBII, TSAb, T4, and T3. This study was the first to use mouse TSHR to induce the disease in mice. Mice were equally susceptible to immunization with either a full-length human TSHR expressed on 293 human cells (xenogeneic cells that are histoincompatible) or purified ectodomain of the human TSHR. This showed that the cells expressing the TSHR need not act as APCs and that all necessary T and B cell epitopes are present on the ectodomain. Similarly, mice immunized with m12 cells (a B lymphoblastoid cell line with H-2D MHC haplotype, same as that of BALB/c mice) and expressing either mouse or human TSHR developed the disease with a similar time of onset, frequency, and severity. In addition, some groups of mice showed a reduction in body weight and thyroid infiltration of lymphocyte. In this model, development of the disease is a slow and progressive process with antibodies against TSHR that can be detected only by ELISA, appearing at approximately 4–6 wk after initiation of antigen administration. This is followed by the appearance of TBII activity by d 60–80 and TSAb by d 150, with clinical onset of the disease by d 250 or later.
Why do the pathogenic antibodies (i.e., TSAbs) develop late in the immune response and result in a delay in the onset of the disease? Although the answer remains uncertain, it is most likely due to the time required for affinity maturation and/or repertoire spreading eventually leading to the production of pathogenic antibodies. Although the reasons for the delay are not known, we speculate that TSAbs might have to interact with TSHR with a relatively higher affinity, and/or with specific epitopes to mediate their TSH agonist activity. It is well known that affinity maturation is largely dependent upon somatic hypermutation of the Ig genes, which in turn is dependent on DNA replication and cell division. This would imply slow and continuous evolution of anti-TSHR antibody responses toward the production of higher affinity antibodies. Alternatively, these results suggest that antibody specificity might gradually spread from one that is restricted against immunodominant epitopes to include cryptic epitopes. This later phenomenon of epitope spreading has been clearly shown in other autoimmune diseases (161, 162, 163, 164). In this scenario, the initial immune response against a self-antigen is mostly directed against the dominant epitopes (operationally defined) and is not pathogenic. However, as the immune response evolves, it may become directed against pathogenic cryptic epitopes. Should immunodominant epitopes become pathogenic, deleterious consequencess would be anticipated. Therefore, for the survival of the species, it is imperative that the pathogenic epitopes remain cryptic. Efforts are under way to use a receptor preparation that has the stimulatory antibody binding sites but is devoid of the immunodominant regions of the receptor, which are irrelevant for TSAb binding or induction, to determine whether the disease can be induced within a shorter duration. Our unpublished studies suggest repertoire spreading as indicated by the change in the TCR Vß and the TSHR peptide specificity of TSHR reactive T cells during the evolution of the immune response. Further studies are required to determine whether both B cell repertoire spreading and somatic hypermutation contribute to the evolving immune response against the TSHR.
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VII. Graves’ Ophthalmopathy |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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; Ref. 165). Although some patients experience only mild ocular discomfort, 3–5% of patients suffer from intense pain and inflammation with double vision or even loss of vision. In addition, a small percentage of patients with GD have clinically apparent pretibial dermopathy, a diffuse or nodular thickening of the skin on the anterior lower leg (Fig. 3). These skin changes occasionally occur on other parts of the body, often after local trauma. Although eye and pretibial skin changes are clinically obvious in the minority of patients with GD, subtle eye and skin changes can be detected using orbital or dermal ultrasonography, or other sensitive detection techniques, in the majority of patients (166, 167). Conversely, although approximately 10% of patients with GO do not have hyperthyroidism, the majority have laboratory evidence of thyroid autoimmune disease, including the presence of antibodies directed against thyroid antigens such as TSHR or thyroid peroxidase (168). Regardless of whether hyperthyroidism occurs first, the signs and symptoms of GO become manifest in 85% of patients within 18 months (169).
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B. Pathological findings
Many of the clinical symptoms and signs of GO can be explained on a mechanical basis by an increase in the volume of both the orbital fatty connective tissues and the extraocular muscle bodies (170). This expanded tissue volume within the orbit in GO can be measured in computed tomography scans taken of GO patients’ orbits (171). Some patients appear to have enlargement of extraocular muscles as the predominant anatomical change. Others exhibit little eye muscle involvement but show significantly increased orbital adipose tissue volume. Histological examination of extraocular muscles reveals intact muscle fibers that are widely separated by edematous connective tissues (172). These perimysial tissues contain excess glycosaminoglycans (GAGs) that appear composed predominately of hyaluronan and chondroitin sulfate (173). A similar accumulation of GAGs, with more profound adipose tissue expansion, is apparent in the fatty connective tissue compartments of the posterior orbit (Fig. 4; Ref. 174). It remains likely that both the accumulation of GAGs and the expansion of the adipose tissues contribute to the increase in orbital tissue volume characteristic of GO. However, further quantitative studies are needed to reliably partition the net effects imposed by hyaluronan accumulation and de novo adipogenesis in GO.
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VIII. Genetic and Environmental Contributions to GO Pathogenesis |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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B. Smoking
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. 187). However, the association between smoking and GO has been shown to be much stronger, representing the strongest risk factor known for this condition. The odds ratio, relative to controls, has been reported to be as high as 20.2 for current smokers and 8.9 for current and ex-smokers, suggesting a direct and immediate effect of smoking (188, 189). In addition, other studies have shown that among patients with GO, smokers have more severe eye disease than do nonsmokers (190).
The mechanisms involved in the association between smoking and GO are unclear. That smoking has been linked to other autoimmune diseases, including rheumatoid arthritis (191) and Crohn’s disease (192), suggests that there may be a generalized stimulation of autoimmune processes in smokers. However, it is doubtful that changes in serum levels of cytokines are responsible, because concentrations do not differ in smokers and nonsmokers, except that smokers do tend to have higher IL-6 receptor (IL-6R) levels (193). In addition, although patients with hyperthyroidism and GO have higher levels of IL-6R receptor than those with hyperthyroidism alone (192, 194), IL-6R does not appear to differ between smoking and nonsmoking GO patients (193). These results would seem to suggest that elevated IL-6R reflects, rather than causes, orbital disease. Thyroid hormone excess per se is unlikely to contribute to the association because other causes of hyperthyroidism or goiter, including toxic nodular goiter, sporadic nontoxic goiter, and Hashimoto’s thyroiditis, are not associated with smoking. Studies in vitro have demonstrated increased GAG production when orbital fibroblasts were cultured under hypoxic conditions (195). In addition, tobacco products enhanced IL-1 secretion by these cells, which in turn has been shown to increase GAG production.
C. Radioiodine treatment for GD
Recent interest has surfaced concerning whether the choice of therapy for hyperthyroidism in GD (131I ablation, surgery, or antithyroid drugs) has an effect on the development or progression of GO. A causal relation between radioiodine treatment and the development of GO is plausible. Such treatment could cause thyroid follicular disruption with the release or new exposure of thyroid autoantigens, especially TSHR, although this has never been shown directly. Indirect evidence of this phenomenon occurring after radioiodine treatment includes T cell activation, prolonged elevation of TSHR antibodies (196), and a transient increase in serum pro- and antiinflammatory cytokines (197).
Several large prospective clinical trials have suggested a direct relationship between 131I treatment and the development or progression of GO (198, 199). The best designed of these studies examined hyperthyroid patients with mild or no GO who were initially treated with a 3- to 4-month course of methimazole (200). Patients were then randomly assigned to receive radioiodine therapy, radioiodine plus prednisone, or continued methimazole therapy. Among the 150 patients treated with radioiodine, ophthalmopathy developed or worsened in 23 (15%) between 2 and 6 months after treatment and improved in none. The worsened eye disease was transient and very mild in 15 of these patients and persistent in eight, seven of whom had evidence of GO before radioiodine treatment. Among the 145 patients treated with radioiodine and prednisone, 67% had improvement, and none worsened. Of the 148 patients treated with methimazole, four patients (3%) had worsening, whereas three patients improved. Although the frequency of worsened GO was significantly higher at 6 months after treatment in the radioiodine group than in either of the two other groups (P < 0.001), this effect was very mild and was not present at 1 yr. The authors concluded that worsening of GO after radioiodine therapy is often transient (and mild) and can be prevented by the concurrent administration of prednisone to selected, high-risk patients. Patients who might benefit from this duo-treatment include those with preexistent GO, smokers, and those having no significant contraindications to corticosteroid therapy (201).
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IX. Orbital Autoimmunity |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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Immunohistochemical studies have demonstrated the presence of several cytokines, including IFN-, TNF-, and IL-1, within the retroocular tissues of patients with severe GO (204). Immunoreactivity for these cytokines was noted both in the cytoplasm of infiltrating mononuclear cells and in the adjacent connective tissue. The preferential expression of these compounds in areas adjacent to mononuclear cell aggregates suggests that these cells are an important source of orbital cytokines. However, in the case of IL-1, marked immunoreactivity was also found in the fatty connective tissues remote from mononuclear cell infiltrates. Thus, it is possible that IL-1 is produced by infiltrating cells and fibroblasts residing within the orbit.
B. Orbital T cell repertoire
A central role for T cells in GD and GO was suggested by several studies demonstrating restriction of TCR V region gene usage in Graves’ thyroid tissue or in orbital connective/fatty tissues and pretibial tissue of patients with active, inflammatory GO (84, 85). The later study also found that orbital tissues of patients with long-standing, inactive GO or with unrelated orbital conditions showed minor or no restriction of TCR V gene usage (205).
Recruitment of T cells with a wide range of specificity (i.e., more divergent sets of expressed TCR genes) appears to follow tissue destruction and cytokine-induced antigen expression occurring later in the disease (206). In this study, comparison of TCR V gene usage between patients showed clear heterogeneity. However, a few striking interpatient similarities in V and Vß gene family usage were noted, especially in Vß2 and Vß6 TCR genes. Taken together, these observations indicate that a limited degree of clonality exists among certain populations of thyroidal, orbital, and pretibial lymphocytes. These findings suggest that the antigen-driven T cell response might be focused during the earlier stages of the immune response in GO. However, later during the disease they exhibit heterogeneity in TCR gene usage. It may be that the important antigen-specific T cells within the orbit undergo activation-induced apoptosis upon contact with the relevant autoantigen and that the T cells remaining in the orbit are nonspecific bystanders.
C. Cytokine production in the orbit
Most cytokines of pathological relevance in disease are produced within the involved tissues. Therefore, measurement of serum cytokine levels may prove irrelevant in GO. Several studies have attempted to characterize the profile of cytokines secreted by orbital-infiltrating T helper (CD4+) cells in GO to determine whether these cells are involved primarily in a cell-mediated (Th1) or a humoral-mediated (Th2) immune response. Two groups of investigators reported that the majority of orbital T cell clones in GD produce Th1-type cytokines IL-2, IFN-, and TNF-, but not IL-4 or IL-5 (207, 208). A third group detected the presence of mRNA encoding a Th2 dominant profile IL-4, IL-5, and IL-10 (209), whereas another group of investigators identified clones secreting cytokines characteristic of subtypes IFN-, IL-4, and IL-10 (210). These studies suggested that T helper cells of both subtypes may be represented in the retroocular infiltrates in GO, perhaps at different times during the course of the disease. This shift in T cell repertoire could result from epitope spreading within the same target antigen or involvement of different antigens. In the absence of information on the antigen specificity of infiltrating T cells, it is very difficult to fully evaluate the relevance of these findings to GO.
In another study, a total of 117 T cell clones were established from orbital adipose/connective tissues of six GO patients, and cytokine production was measured in 57 CD3+CD4+ clones (211). Th1-Type clones were found to predominate in cultures from patients with recent onset (<2 yr) of hyperthyroidism (Th1/Th0/Th2 = 57/29/14%) or GO (Th1/Th0/Th2 = 47/30/23%). In contrast, Th2-type clones predominated in cultures from patients with more remote onset (>2 yr) of hyperthyroidism (Th1/Th0/Th2 = 0/31/69%) or GO (n = 4; Th1/Th0/Th2 = 0/25/75%). Although the CD3+CD4+ clones characterized were not necessarily tissue-antigen-specific, these findings suggest that cell-mediated (Th1-type) immune reactions may predominate in the orbit in early, inflammatory stages of the disease. The proinflammatory cytokines (IFN-, IL-2, and TNF-) produced by these cells, along with IL-1 derived primarily from macrophages and fibroblasts, may prove responsible for stimulation of GAG synthesis in orbital fibroblasts and might function as mediators of inflammation in early disease. In addition, the CD40/CD154 could serve as an important activational bridge between T cells and orbital fibroblasts. These same cytokines stimulate the expression of immunomodulatory molecules (HLA-DR, ICAM-1, and HSP-72) on orbital fibroblasts, potentially enhancing the propagation of autoimmune responses within the orbit (166). In the recovery phase of GO, T helper type 2 lymphocytes (producing IL-4, IL-5, and IL-10) could play an important role and may mediate the late-stage fibrosis of the extraocular muscles (212).
D. Cytokine effects in the orbit
Cytokines (213, 214) and chemokines (210) are capable of inducing the expression of immunomodulatory proteins in orbital fibroblasts and could contribute to the propagation of the disease process. In addition, regulated on activation, normal T cells expressed and secreted (RANTES), IL-16, and monocyte chemoattractant protein-1 may be important in triggering T cell migration across activated microvascular endothelium into the orbit. IL-1, TGF-ß, IGF-I, IFN-, leukoregulin, and coculture with mast cells have been shown to stimulate accumulation of GAGs in orbital fibroblast cultures (213, 214, 215, 216, 217). Preliminary studies suggest that some cytokines can either inhibit (TGF-ß, IFN-, and TNF-) or stimulate (IL-6) adipogenesis in cultures of orbital fibroblasts (218, 219). As will be discussed later in this review, enhanced expression of the TSHR within the orbit may play a role in the initiation or propagation of the autoimmune response in GO.
Cytokines are also capable of inducing the expression of several proteins in orbital fibroblasts, including HLA-DR, intracellular adhesion molecule-1, and heat shock protein-72 (166). If reflective of events occurring in vivo, their enhanced expression in inflammatory states within the orbit would be expected to influence the ongoing autoimmune response. Another effect of cytokines relevant to GO is their ability to stimulate the proliferation of orbital fibroblasts. Significantly increased proliferation was observed after treatment with IL-1, IL-4, IGF-I, and TGF-ß, but not with IL-2 or IL-6 (220). This effect of cytokines on orbital fibroblasts may be contributory in the later stages of GO when fibrosis of the extraocular muscles may result in debilitating double vision.
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X. Orbital Autoantigens |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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A. Target cells and candidate autoantigens
Much effort has been directed at identifying target cells and mapping the critical epitope on autoantigens they display. The presence of gross muscle enlargement had led early investigators to use eye muscle. In these studies, sera from GO patients were used as probes in enzyme-linked immunosorbent assays (221, 222, 223). These studies involved crude preparations of soluble nonhuman eye muscle as the source of antigen, due to difficulties inherent in culturing muscle cells and a lack of available human eye tissue. Although the presence of circulating antibodies directed against various eye muscle preparations was reported, the findings were not confirmed by other investigators or the antibodies were subsequently found to be nonspecific (224). Immunoblots demonstrated serum reactivity against a 64-kDa eye muscle membrane antigen (225, 226) and a 23-kDa orbital fibroblast antigen (227). The eye muscle membrane antigen was recognized by approximately 70% of GO sera, especially those sera from patients with active or recent disease, and appeared to react with a protein in the thyroid but not in skeletal muscle. The 23-kDa protein was found to be cytosolic, detected in cultured orbital fibroblasts from patients with GO; it was recognized by 56% of IgG class antibodies in sera from patients with GD, but with no distinct bias in favor of the subgroup with GO, compared with 15% of controls. Because neither the eye muscle membrane antigen nor the orbital fibroblast antigen was found to be restricted to orbital tissue and the antibodies were detected frequently in normal individuals, it is not likely that these antibodies are directly involved in GO pathogenesis.
The screening of a 
gt11 human thyroid cDNA library with a pool of Hashimoto’s thyroiditis sera led to the isolation of a clone termed D1, encoding a 97-amino acid peptide (228). The full-length cDNA was found to encode a 63- to 64-kDa protein that was thought to be the same antigen as identified in earlier immunoblotting studies (229). However, this could not be confirmed with peptide sequencing or an analysis of amino acid composition. This protein was expressed 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 mRNA in a wide variety of tissues including uterus, spleen, and parathyroid (230). Furthermore, antibodies to D1 were found frequently in the sera of normal individuals, and their presence was not correlated with the presence of clinical GO in Graves’ patients. Attempts to identify this antigen using gel separation and microsequencing suggested that it might be either calsequestrin, a calcium-binding protein in the sarcoplasmic reticulum of striated muscle (231), or the flavoprotein subunit of mitochondrial succinate dehydrogenase (232). In subsequent studies by the same investigators, a novel protein, termed G2 s, was cloned from an eye muscle gt11 library using an affinity-purified anti-55-kDa protein antibody from a patient with GO (233). However, due in part to very low titers of relevant serum antibodies it proved very difficult to clone and identify a cDNA encoding this protein. Other investigators have concluded that these antigens represent widely expressed cytoskeletal proteins. The inflammatory process occurring within the orbital tissues in GO would lead to tissue destruction with release of these sequestered proteins. Serum antibodies directed against these antigens would be unlikely to play a primary role in GO pathogenesis.
Other candidate autoantigens studied in relation to GO include acetylcholine receptor and thyroglobulin. The former was found in extraocular muscle membrane preparations to be recognized by antibodies from GO patients (234). Subsequent studies, in which a human thyroid cDNA library was screened with polyclonal acetylcholinesterase antibodies, led to the isolation of two thyroglobulin segments having close homology with acetylcholinesterase (235). These findings were especially interesting in light of earlier studies in which thyroglobulin or a related protein was detected in the orbit by immunohistochemistry, and possible connections between the thyroid and orbit were shown using radioisotope-based lymphography (236). Investigators hypothesized that these connections might allow transfer of thyroglobulin into orbital tissues (237). However, the potential significance of this finding is unclear, especially because patients with GO don’t have particularly elevated antithyroglobulin antibodies, and patients with Hashimoto’s thyroiditis generally don’t have evidence of GO. Weightman et al. (238) showed that 125I-IGF-I binding to the surface of orbital fibroblasts was displaced by IgG from patients with GD, suggesting that antibodies directed against the IGF-I receptor or IGF-I binding proteins might be present in the serum.
B. TSHR
As discussed elsewhere in this review, the autoantigen involved in the hyperthyroidism of GD is the TSHR. The close clinical relationships between GD, GO, and pretibial dermopathy make this protein a good candidate autoantigen for involvement in the orbital and dermal manifestations of the disease. Several studies have examined the correlation between the presence or level of TSHR antibodies and the severity of GO with conflicting results. The most carefully designed of these studies found both thyroid stimulatory Igs (TSI) and TBII to be closely correlated with a GO clinical activity score (239). Weaker but significant correlation was also noted between antibody levels and proptosis. Several studies, with conflicting results, have examined the relationship between TSHR antibody titer and severity of GO. In GD, stimulatory TSHR antibodies cause hyperthyroidism. The titers of these antibodies may reflect the severity of thyrotoxicosis and degree of TSHR-specific lymphocytic activation (240). Similarly, because levels of TSHR antibodies may reflect GO clinical activity, it may follow that infiltrating T cells found within the orbit in GO specifically recognize TSHR expressed in those tissues. The TSHR-directed antibodies produced subsequently, either locally or in the peripheral lymphatic tissues (i.e., lymph nodes and spleen), however, may not have a direct pathogenic role within the orbit in GO but may rather reflect the intensity of the orbital autoimmune response. However, whether TSHR or autoantibodies directed against it are involved in pathogenesis remains uncertain.
Studies aimed at determining whether TSHR is present in normal or GO orbital tissues or cell cultures led to reports by several laboratories identifying TSHR mRNA, or a variant TSHR transcript, in various orbital preparations using the RT-PCR (241, 242, 243). However, RNA transcripts detected only by PCR-based amplification of cDNA may have little physiological relevance. To clarify this issue, further studies were performed using a ribonuclease protection assay for semiquantitative detection of this low abundance mRNA (244), Northern blotting (245) for mRNA detection, or immunohistochemistry (246) for TSHR protein detection. These studies documented the presence of mRNA and protein in GO orbital adipose/connective tissue specimens. In contrast, TSHR either was not detected or was found to be expressed at low levels in normal orbital fatty connective tissue samples and derivative cultures. However, these studies failed to examine samples of disease-derived and control tissues from a large number of different donors under standardized conditions and therefore were probably not quantitative. In addition, it should be noted that TSHR mRNA has subsequently been detected in a number of different tissues, including those not ordinarily involved in GD. Thus, additional studies will be necessary to confirm that TSHR is a target autoantigen expressed at higher levels in orbital tissues.
It is possible that TSHR is constitutively expressed at very low levels in extrathyroidal tissues in normal individuals, including orbital tissues, skin, and other areas not usually clinically involved in GD. This expression may be increased in GD after stimulation by unknown factors, including particular inflammatory cytokines. Alternately, orbital TSHR expression may be enhanced secondarily as a consequence of orbital adipogenesis, which is increased in GO. In any event, increased local TSHR expression could make the receptor available to act as an autoantigen within the orbit, the pretibial skin, or other anatomic regions occasionally involved in the disease. The presence of systemic subclinical connective tissue inflammation in GD, as has been suggested, might predispose to this series of events. Additionally, local factors, such as gravitational dependency, trauma, cigarette smoking, or anatomic constraint of the bony orbit, may lead to clinical disease involvement at specific extrathyroidal sites (247).
Evidence that orbital adipogenesis, with accompanying TSHR expression, may be involved in GO has been generated from cell culture studies of orbital preadipocyte fibroblasts (248, 249). These results obtained using human orbital fibroblasts are consistent with observations made in rodent fibroblasts (250, 251, 252). Preadipocytes are cells of the fibroblast lineage that can, under certain culture conditions, differentiate into adipocytes. Sorisky et al. (248) demonstrated that orbital fibroblasts contain a subpopulation of cells that behave like preadipocytes when incubated with cAMP-enhancing compounds and prostacyclin. Initial studies disclosed that 5–10% of the cells from a typical parental strain of orbital fibroblasts undergo differentiation (248). Subsequent studies from this same group have demonstrated that approximately 50% of the cells undergo this differentiation when subjected to medium-containing ligands of the adipogenic trigger, peroxisome proliferator-activated receptor- (PPAR-) (249). Moreover, the adipocytes fail to express surface Thy-1. Very recent studies suggest that the subsets delineated in orbital fibroblasts by display of Thy-1 define discrete functional populations of cells. Thy-1+ cells fail to differentiate into lipid-bearing adipocytes but when treated with TGF-ß develop into myofibroblasts and express relatively high levels of smooth muscle-specific actin (Koumas, L., T. J. Smith, and R. P. Phipps, unpublished observations). In addition to undergoing adipogenesis, orbital cells show enhanced TSHR expression when cultured under these conditions (249). These findings suggest that there may be a humoral stimulus for adipogenesis and TSHR expression present either systemically or within the orbit in GO. The cytokine IL-6 has been shown to enhance adipogenesis and TSHR expression in cultured orbital preadipocyte fibroblasts (219). The cytokine has been shown to be elevated in the sera of patients with GD and GO (253, 254). The cellular source of IL-6 is uncertain. But it is of potential interest that IL-6 is expressed by orbital fibroblasts and thyrocytes challenged with cytokines such as CD40 ligand (255, 256, 257). It is possible that IL-6 serves as an important mediator of these changes occurring within the orbit in GO.
Studies concerning the pathogenesis of GD have long been hampered by the lack of a useful animal model. Recently, a novel animal model was developed in which thyroiditis was transferred to naive BALB/c mice with splenocytes that were primed with either TSHR fusion protein (136) or the cDNA for the human TSHR (258). Thyroiditis was induced in the majority of these animals and was associated with the production of low-titer TSHR antibodies, although the majority of antibodies were TSH binding inhibiting, rather than stimulatory, Igs. Examination of the orbits in 17 of 25 of these animals revealed lymphocytic and mast cell infiltration, accumulation of adipose tissue and edema with material staining with PAS, dissociation of muscle fibers, and evidence of TSHR immunoreactivity (257). Whether this particular animal model accurately reflects the autoimmune processes involved in the development of GD is unclear. In fact, in another study, vaccination with the naked TSHR elicited a Th1 T cell response in which IFN-, rather than autoantibody, production dominated the immune response (258). Although falling short of clearly exhibiting the spectrum of fully developed GD or demonstrating a primary role for TSHR as an orbital target antigen, these models potentially "open the door" for more robust models of the disease.
C. Orbital T cell reactivity
Direct evidence that any particular candidate antigen or orbital cell type is an autoimmune target in GO has been difficult to obtain, largely due to difficulties in obtaining orbital lymphocytes and autologous orbital tissues from GO patients. However, several studies have examined reactivity of orbital T cell lines against various purified antigens or orbital tissue preparations. In one report, CD8+ T cell lines recognized autologous cultures of orbital fibroblasts in an MHC class I-restricted manner (259). These T cells also proliferated in response to thyroid membrane preparations and purified TSHR. No proliferation in response to crude eye muscle extract, autologous peripheral blood mononuclear cells, allogeneic fibroblasts, or purified protein derivative of mycobacterium tuberculosis was noted. Another group of investigators found that orbital and peripheral T cell lines from patients with GO proliferated in response to autologous orbital fibroblast-derived proteins in the 6- to 10-kDa and 19- to 26-kDa range but did not recognize proteins derived from autologous orbital myoblasts or abdominal adipose or muscle tissue (260). Candidate antigens, including recombinant D1 and thyroglobulin, were tested in another set of experiments (261). Although no T cell responses to these antigens were observed, cells from some patients exhibited weak response to eye muscle proteins between 25 and 50 kDa. In contrast, orbital fibroblasts induced striking proliferation of circulating T cells from some patients.
The interpretation of these studies is limited, in part, by 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 recombinant proteins, as the antigens precluded precise antigen identification. Studies of endogenously processed and presented antigens would be particularly useful in this regard. Such studies would no doubt facilitate an understanding of immune responses involved in the development of GO.
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XI. Role of Orbital Connective Tissue in GO |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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XII. Role of GAGs in GO |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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Hyaluronan is a high molecular weight molecule and is a member of a group of complex carbohydrate molecules called GAGs (262). GAGs are linear polymers with characteristic physicochemical properties. They are large, bulky, polyanionic compounds composed of repeating disaccharides, each of which comprises an amino sugar and a uronic acid residue. One molecule of hyaluronan with a molecular weight of 106 Da possesses a spherical diameter of 4,000 Å and a volume of 330,000 x 10-19 ml. An equivalent weight of hyaluronan occupies a volume that is 75,000 times greater than that of collagen. When covalently linked to core proteins, complexes of GAG are known as proteoglycans. Among the distinct molecular characteristics displayed by GAGs are rheological properties that underlie their extremely hydrophilic nature and their viscosity in solution. Hyaluronan differs from the other abundant GAGs in that it does not contain a core protein and it is not sulfated, unlike chondroitin, dermatan, and heparan sulfates. It would appear that of these molecules, hyaluronan is accumulating predominately in connective tissue lesions of GD. It is composed of alternating residues of N-acetylglucosamine and D-glucuronic acid (262). The repeating sugars form a basic disaccharide organization with alternating linkages. Hyaluronan synthesis occurs at the plasma membrane where the alternating sugar residues are transferred sequentially from their respective uridine diphosphate (UDP) donors by hyaluronate synthases (270) (Fig. 6). This enzyme activity localizes to the membrane fraction of disrupted oligodendroglioma cells in culture. Great progress in defining the molecular biology of hyaluronan synthesis has been made over the past few years. A family consisting of three hyaluronan synthase (HAS) isoforms has been identified recently, and all members have been cloned (271, 272, 273). The tissue distribution of the HAS enzymes appears to differ, as does the putative role that each might play in normal development and in the pathogenesis of human disease. The expression of all three HAS transcripts has been shown to be up-regulated by IL-1ß in cultured human orbital fibroblasts (219). In addition, the enzyme immediately upstream from the HAS enzymes, termed UDP-glucose dehydrogenase (UDP-GD), has also been characterized (274). This enzyme catalyzes the conversion of UDP-glucose to UDP-glucuronate. UDP-GD is induced substantially by proinflammatory cytokines in orbital fibroblasts, and this induction can be attenuated by physiologically relevant concentrations of glucocorticoids (274). It is unclear whether, in the case of GD, hyaluronan synthesis is accelerated or whether macromolecular disposal might be delayed. Hyaluronan degradation is catalyzed by a group of enzymes called hyaluronidases. Depending on their tissues of origin, they exhibit distinct profiles of substrate specificity. Bacterial hyaluronidases, such as those expressed by Streptomyces, are extremely specific for hyaluronan (275). They are therefore particularly important tools for the biochemical analysis of GAGs. Whereas animal fibroblasts express hyaluronidases, those from human skin and orbit fail to produce hyaluronan degrading enzymes (276, 277). These differences in hyaluronidase expression found in fibroblasts from different sources may reflect interspecies variations or may indicate loss of a particular phenotypic attribute in human fibroblasts. Although GAGs function in the extracellular compartment, they may need to be transported into lysosomes for complete degradation. Human skin fibroblasts are incapable of internalization of hyaluronan (278), unlike other cell types, and this may explain their inability to degrade the macromolecule completely. Because human fibroblasts do not appear capable of degrading hyaluronan, it is essential that other cell types might express hyaluronidases in vivo, the human orbit. Alternatively, hyaluronan disposal could occur through relatively nonspecific pathways.
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Many of the consequences of hyaluronan accumulation in the orbit result from material bulk. In addition, low molecular weight hyaluronan has been shown to influence gene expression of cells exposed to the complex carbohydrate (279). In particular, the potential for hyaluronan to induce proinflammatory genes in immunocompetent cells could lead to substantial amplification of the inflammatory response. Many of the actions of hyaluronan on target cells are mediated through its binding to CD44, which is displayed on the cell surface and is competent to signal downstream events through the activation of kinase cascades (280). Another hyaluronan binding protein/receptor of potential importance is RHAMM (281). This protein, first described by Turley and colleagues (281), binds with high affinity to hyaluronan. That group contends that hyaluronan binds to RHAMM and in so doing stimulates cell locomotion. Moreover, this effect may be mediated through rapid and transient protein tyrosine kinase signaling (281). Although the function of this second receptor is less well-defined than that of CD44, binding of hyaluronan to target cells through RHAMM may lead to changes in cell biology and gene expression (282). Emerging as an important direction for future investigation is a clearer definition of the molecular events underlying hyaluronan accumulation and the biological consequences of a disordered economy of the complex sugar in GO.
B. Orbital fat expansion and muscle enlargement can contribute substantially to tissue remodeling in GO
Although most individuals with GO present with evidence of both muscle enlargement and increased volume of the fatty depot in the orbit, some patients appear to exhibit a predominance of either (283). Those individuals younger than 40 yr are considerably more likely to manifest orbital fat expansion-related proptosis in the absence of muscle infiltration (283), whereas older patients, more than 70 yr old, can develop severe, isolated muscle enlargement associated with compressive optic neuropathy (284). This presentation can be attributed to fusiform enlargement of the extraocular muscles. When assessed early in the course of the disease, the contractile elements of the muscle remain intact in those few patients in whom such an examination has been possible. It would appear that the connective tissue investments surrounding the muscle itself are altered (285). Specifically, there is accumulation of GAGs and infiltration of the perimysium with mononuclear cells, including T lymphocytes and mast cells.
Orbital fat has been the topic of investigation for many years. The elegant, pioneering work of Smelser examined the fat of animals in which experimental ophthalmopathy had been induced by first thyroidectomizing guinea pigs and then injecting the animals with extract of the anterior pituitary (265). The pituitary extract produced a 35% increase in the weight of the orbital contents. Only the ventral lacrimal gland was spared. The fat of the orbit nearly doubled in weight and acquired a different appearance; it became gelatinous and translucent rather than opaque as in the control animals. Edematous fluid appeared to have infiltrated between fat cells. The edematous material stained strongly with aniline blue or eosin but failed to stain like amyloid or mucoid material.
Orbital fat has been examined in the context of GO using multiple imaging modalities. Measurement abnormalities were found in 87% of patients with GD with clinically detectable GO. In contrast, 70% of hyperthyroid patients without clinical signs were found to have abnormal pixel-calibrated volume measurements of muscle and fat by CT scans in a study involving 72 subjects (171). Enlargement of fat in addition to muscle was documented in 46% of patients and isolated volume expansion of fat in 8%. In another study, 15 patients presenting with proptosis but without masses or muscle enlargement were studied, and four were found to have GO (285).
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 exposed. In general, it has become evident that adipogenesis is a tightly regulated process and that key molecules initiate the differentiation of preadipocytes to mature fat cells. Candidate molecules include natural ligands of PPAR- (286, 287). The best characterized of these are prostaglandins (PGs) of the PGJ2 series and related prostanoids. Unfortunately, no information currently exists concerning PG production or tissue levels in orbital tissue in situ. Moreover, whether GO is accompanied at least in some patients with a shift in the homeostatic mechanism governing orbital fat mass is not known. Of potential relevance to the issue of enhanced adipogenic differentiation in GO is the finding of fatty infiltration in extraocular muscle (280). Although no stringent quantitative analysis of hyaluronan content in orbital tissues currently exists, several qualitative observations suggest that the GAGs accumulate in both muscle and connective tissues. Thus, the increase in fat cell number and or size may contribute to fat expansion in GO (288). But the contribution of infiltrating GAGs to this process has not been quantified.
Much of the insight into cellular infiltration of the orbital tissues in GO derives from histological examination relatively late in the course of the disease. The paucity of information causing early GO results from a lack of access to tissues before the end stages of the disease. There exist few indications for surgical intervention during the active phases. Consequently, we do not know the identity of the initial immunocompetent cell types recruited to the orbit that share proximate responsibility for the earliest processes. These cells orchestrate the inflammatory response and initiate tissue remodeling. Rather, the relatively few reports concerning infiltrating cells in GO appear to define the reactive components of the disease. Although T cells dominate the histological picture of late GO, their prominence in many examples of dermopathic skin is less obvious. In addition to lymphocytes, mast cells are often abundant (174). This may have substantial importance because of the role that these cells play in the genesis of fibrosis. Both T cell phenotypes are present in orbital lesions late in the disease (258, 289, 290), but whether CD4+ or CD8+ cells are dominant early remains controversial. A number of cytokines have been detected in the orbital tissues in GO. Among these are TNF-, IL-1, IFN-, and TGF-ß (207). These studies have relied largely on RT-PCR-based assays attempting to quantify cytokine mRNAs, or they used immunostaining. Although the reports have provided the first approximation of the cytokine milieu found in GO, they are neither specific nor quantitative. Moreover, important and direct comparisons between the cytokine profiles found in GO and other types of orbital inflammatory diseases have yet to be conducted. These and more quantitative studies will be necessary to define those molecular triggers present in the earliest stages of pathology that might distinguish GO from other forms of orbital inflammation. The lack of robust animal models for GO has also proven an important limitation in defining the cellular and biochemical events that provoke initiating events. The orbital manifestations contained in most reports of experimentally induced rodent disease have been disappointing (257, 291).
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XIII. Role of Orbital Fibroblasts in GO |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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Recent evidence has been advanced to support the concept of fibroblast diversity. Fibroblasts from certain anatomic regions appear to differ from those found elsewhere in the human body. Moreover, within certain tissues, subpopulations of cells can be identified. Discrete subsets of fibroblasts can now be identified on the basis of expression of cellular markers such as surface proteins, carbohydrates, and gangliosides. Some of those subpopulations appear to represent functional subsets. Identification of cells within a tissue that behave differently suggests that the physiological roles of subsets in health and disease may diverge. This phenomenon may resemble the situation concerning lymphocytes, where cells are distinguished on the basis of surface determinants and marked functional differences among them have emerged. Thus, the realization that discrete fibroblast subsets populate some tissues represents an important insight into the complexity of tissue organization. The pioneering observations concerning fibroblast subsets within a tissue were made by Phipps and his colleagues (293). They discovered that some fibroblasts from the murine lung fail to express and display surface Thy-1. This determinant represents a surface glycoprotein, the natural ligand of which has yet to be identified (294). Thy-1 has been shown recently to bind integrin ß3 on astrocytes and, in so doing, to trigger the tyrosine phosphorylation of focal contacts (295). Phipps et al. (296) determined that the surface display of Thy-1 by murine lung fibroblasts defined cells with several phenotypic attributes that appeared divergent. For instance, only Thy-1- fibroblasts display class II major histocompatibility complex antigens after stimulation with IFN- (296). When activated with TNF, only the Thy-1- fibroblast subset expresses IL-1 (297). In contrast, Thy-1- fibroblasts failed to express either IL-1 or IL-1ß. Thy-1+ fibroblast subsets produce substantially more collagen in vitro, suggesting that these cells might have particular importance in the pathogenesis of fibrosis (298). On the other hand, IL-6 appears to be expressed in both Thy-1+ and Thy-1- subsets (299). Moreover, the expression of immunologically important molecules, such as PGs, appears to differ in the two fibroblast subsets. Thus, precedent exists for subsets exhibiting very distinct phenotypic characteristics that would be potentially important for how they might participate in inflammatory responses and fibrosis. Korn and his colleagues (300, 301, 302) have demonstrated substantial clonal variations with regard to PGE2 synthesis among synovial fibroblasts. These differences among subsets were stably expressed through many population doublings and substantial time in culture. Moreover, they were not restricted to a particular stimulus. If the pattern of fibroblast behavior in synovial cultures is relevant to those from orbit, one would expect similar profiles of PGE2 production in those cells.
B. Orbital fibroblasts are different from many other types of fibroblasts
Human fibroblasts have been examined rather extensively in culture in an effort to understand their potential role in the pathogenesis of GD. In particular, cultures derived from the skin of the anterior leg and the orbits have gained substantial attention, largely because the lesions affecting connective tissue in GD occur most frequently in those areas. Among the first studies involving cultured orbital fibroblasts were those of Sisson and colleagues (303, 304, 305). Sisson characterized the GAG synthesis in orbital fibroblast cultures derived from the connective tissues. Those studies revealed that orbital fibroblasts exhibited a substantial response to lymphocytes with regard to hyaluronan synthesis glucose utilization (304). Sisson and Vanderburg reported that glucocorticoids failed to inhibit basal hyaluronan synthesis in orbital fibroblasts but could block lymphocyte-provoked GAG synthesis in those same cells (305). A more recent report demonstrated a similar relative insensitivity of hyaluronan synthesis in orbital fibroblasts to the actions of T3 and dexamethasone (277). Hyaluronan synthesis in dermal fibroblasts has been shown to be inhibited substantially by physiological concentrations of glucocorticoids (306) and thyroid hormones (307). Whether orbital fibroblasts are insensitive to these hormones as a consequence of differences in receptor complement or postreceptor signaling is uncertain.
Orbital fibroblasts, like those derived from certain other anatomic regions, exhibit phenotypic attributes that set them apart. They have a distinct morphology in vitro (277). Orbital fibroblast cultures contain both angular (stellate-shaped with three or more cytoplasmic processes) and fusiform (spindle-shaped with two or three dendritic processes) cells (277). The perinuclear cytoplasm contains prominent granular features. Dermal fibroblasts examined at the same time contain predominantly angular cells with a more homogeneous and less granular cytoplasm. In contrast, the ultrastructure of orbital and nonorbital fibroblasts is remarkably similar, as determined by transmission electron microscopy (308). Orbital fibroblasts display receptors (309) and gangliosides (310) and generate macromolecular components of the ECM differently than do other fibroblasts (214, 311). Using two-dimensional protein gel electrophoresis, Young et al. (312) have systematically compared the basal and cytokine-provoked protein expression in orbital fibroblasts and compared this profile with those of the pretibial and abdominal wall fibroblasts. Notable in these studies was their use of multiple strains from a single donor. They found that, although the vast majority of protein spots were expressed at similar levels of abundance in all three sites and responded the same way to cytokine treatment, a limited number of protein inductions and repressions were restricted to orbital and/or pretibial fibroblast strains (312). The substantial differences between orbital and nonorbital fibroblasts suggest that the former may play unusual if not unique roles in the normal function of the orbit. Plasminogen activator inhibitor type 1 is a key regulator of the pericellular proteolytic environment. When orbital fibroblasts are treated with IFN- (313) or leukoregulin (314), plasminogen activator inhibitor type 1 is dramatically induced. In contrast, expression in dermal fibroblasts is either down-regulated or modestly enhanced. This suggests that in the context of an inflammatory response, orbital fibroblasts exhibit a differentially attenuated proteolytic environment that would favor the accumulation of ECM macromolecules. This difference may underlie some of the qualitative tissue differences found in GO.
It would appear that the population of orbital fibroblasts can be further separated on the basis of specific phenotypic attributes. For instance, approximately 50% of parental strains of orbital fibroblasts display Thy-1 on their cell surfaces (315). Smith et al. (316) reported that separating cells into pure Thy-1+ and Thy-1- cultures led to subsets that faithfully maintained their status over many population doublings and passages in vitro. When segregated, Thy-1+ cells produce more PGE2 and express higher levels of PG endoperoxide H synthase (PGHS)-2 upon treatment with IL-1 or CD154 (317). On the other hand, Thy-1- fibroblasts express much higher levels of IL-8 when activated with proinflammatory cytokines (317). Parental strains of orbital fibroblasts contain preadipocytes that, when subjected to a differentiation protocol, undergo terminal differentiation into fat cells (248). The medium prompting adipogenesis contained cPGI2, insulin, dexamethasone, TSH, and isobutylmethylxanthone (312). Approximately 5% of the cells differentiate into cells that accumulate inclusions staining positively with Oil Red O. It was subsequently demonstrated that Thy-1- subset exhibits substantial adipogenic potential and can differentiate in vitro into mature adipocytes when incubated with ligands of PPAR- (316). Such an adipogenic differentiation in orbital fibroblast cultures has been associated with increases in the levels of expression of the TSHR (249). Unfortunately, those reports contained data that were not convincing. The magnitude of the effects ascribed to differentiation with regard to TSH-provoked cAMP generation was inconsistent, and the usual indications of result variability were missing in several studies. Thy-1+ cultures may not possess the potential to differentiate into fat cells. The demonstration of adipogenic potential in orbital fibroblasts has substantial implications concerning the pathogenesis of GO. Expansion of orbital fat is a prominent feature of the disease associated in some patients with GO (284). Clearly, the full spectrum of cellular differences imposed by the bimodal distribution of Thy-1 on orbital fibroblasts from connective/adipose tissue has yet to be appreciated but could define discrete functional characteristics of each subset. In contrast, fibroblasts associated with and derived from the extraocular muscles display Thy-1 uniformly (317). Like dermal fibroblasts, those from the perimysial connective tissue are resistant to these differentiation protocols (317). This raises the possibility that Thy-1+ fibroblasts can be terminally differentiated into other types of cells such as myofibroblasts. A number of reports have appeared to suggest that fibroblasts exposed to TGF-ß can be induced to differentiate into cells possessing a phenotype associated with myofibroblasts. These cells express high levels of smooth muscle-specific actin. In this regard, fibroblasts lacking adipogenic potential could represent the cells involved in fibrosis and respond to Th2 cytokines, including IL-4, IL-5, and IL-13. These cytokines are associated with robust fibrotic tissue responses. The increases in muscle and fat volumes associated with GO could, at least in part, result as a consequence of tissue expansion from additional muscle and adipocytes differentiating from the respective fibroblast compartments. These apparent differences between fibroblasts from the muscle and fat depots might help explain the diverging clinical presentation seen in old and young patients with GO (318).
C. Orbital fibroblasts express high levels of inducible cyclooxygenase and produce extremely high levels of PGE2 when activated by proinflammatory cytokines
Orbital fibroblasts exhibit exaggerated responses to proinflammatory signals such as those conveyed by cytokines. For instance, leukoregulin, a T cell-derived cytokine, and IL-1 dramatically up-regulate several genes that are relevant to inflammation. These include PGHS-2 (EC 1.14.99.1), the inflammatory cyclooxygenase (319, 320). When PGHS-2 is induced in orbital fibroblasts derived from either normal or GO connective tissue, a dramatic increase in PGE2 synthesis occurs (319). The magnitude of this increase, provoked by a number of cytokines, is considerably greater than that observed in dermal fibroblasts (319, 320). The disparity in levels of PGHS-2 induction may relate to the relatively modest levels of IL-1 receptor antagonist expression observed in cytokine-activated orbital fibroblasts (320). The levels of PGHS-2 mRNA and protein achieved after cytokine treatment are proportionately greater than those in other fibroblasts, and the vast majority of the increased PGE2 production observed is inhibited by PGHS-2 selective inhibitors such as SC58125 (319). This finding suggests that PGHS-2 selective inhibitors, now in the marketplace, might represent effective and safe alternative therapeutics for acute orbital inflammation in GO.
The induction of PGHS-2 requires the activation of the MAPK pathways, including both ERK 1/2 and p38 (321). Moreover, nuclear factor (NF)-
B, a centrally important transcription factor in inflammation-related gene induction, appears critical to the activation of the PGHS-2 promoter in orbital fibroblasts (320). Thus, it would appear that the exaggerated induction of PGHS-2 in orbital fibroblasts helps define the inflammatory phenotype of these cells. In contrast, PGHS-1 are a constitutively expressed enzyme that is abundant in unprovoked fibroblasts (319). Moreover, leukoregulin and IL-1 fail to alter the levels of PGHS-1 mRNA or protein. Although the expression and cytokine induction of PGHS-2 are completely attenuated by physiological concentrations of glucocorticoids, PGHS-1 levels are unaltered by the steroids.
Another enzyme in the PGE2 pathway is also induced by proinflammatory cytokines in orbital fibroblasts (321). Microsomal PGE2 synthase (mPGES; EC 5.3.99.3) is the terminal catalytic determinant in the synthetic cascade for PGE2. mPGES is a glutathione-dependent enzyme that is usually expressed at low levels under unstressed cellular conditions. mPGES is substantially induced by IL-1ß in these cells in a well-coordinated manner with PGHS-2. Induction of the two enzymes by IL-1 shares utilization of the MAPK pathways and involves the modest up-regulation of respective gene promoter activities. PGHS-2 mRNA is extraordinarily labile under basal culture conditions in human orbital fibroblasts due to the presence of at least 22 AUUUA instability elements in its 3' untranslated region. When cells are treated with IL-1ß, the stability of PGHS-2 mRNA is dramatically enhanced. The half-life increases from 1 h to greater than 5 h. This augmentation in the half-life of the PGHS-2 transcript is critical to the up-regulation of the expression of the enzymes. In contrast, mPGES mRNA is extremely long-lived under basal conditions, and IL-1ß does not influence its survival appreciably (321). We hypothesize that the capacity of orbital fibroblasts to produce PGE2 is the basis, at least in part, for the susceptibility of the orbit to inflammatory processes and the tissue remodeling seen in GO. PGE2 exerts an important influence on the nature of immune responses. For instance, the prostanoid biases the commitment of naive T cells Th0 away from the Th1 phenotype and toward that of the Th2 (322). PGE2 plays an important role in the behavior and apoptosis of B lymphocytes (323) and influences mast cell function (324). Thus, the finding that orbital fibroblasts generate particularly high levels of PGE2 when activated suggests the potential of those cells in defining the quality of tissue remodeling found in inflammatory disease of the orbit, such as occurs in GO. The result of this bias can substantially skew the cytokine microenvironment toward profibrotic factors and away from those associated with acute inflammation (325).
D. Fibroblasts are sentinel cells capable of initiating lymphocyte recruitment and tissue remodeling
Fibroblasts from a wide array of human tissues have been shown to express chemoattractant activities when activated by cytokines. The role of recruiting leukocytes to areas of inflammation and tissue damage falls, in some measure, to a family of cytokines called chemokines. These are small polypeptides that range in size from 7 to 10 kDa and comprise different families, depending on their amino acid sequences. They are designated according to the cysteine residue signatures they contain and are divided into three families. Chemokines bind to high-affinity receptors expressed on the surfaces of target cells. The various members of the three families exhibit diverse specificity with regard to the receptors they utilize and the target cells they activate. In addition to chemokines, fibroblasts express other small molecules that lack requisite cysteine signatures but possess activities that prompt leukocytes to infiltrate areas of tissue damage. IL-16 is such a chemoattractant molecule. It binds exclusively to CD4 and therefore only influences the migration of cells displaying CD4 on their surface (326). IL-16 is synthesized as a 69-kDa precursor molecule and is secreted as a 56-kDa protein composed of four identical subunits. The release of IL-16 protein from CD8+ T cells and fibroblasts is dependent on the activity of caspase-3, a cysteine protease (327). At the cell target, IL-16 induces the expression of IL-2 receptors and influences IL-2-dependent cell activation. IL-16 has been implicated in a number of human autoimmune diseases, including rheumatoid arthritis (328), inflammatory bowel disease (329), and lupus erythematosis (330). The other abundant chemoattractant synthesized by cytokine-activated fibroblasts is RANTES, a c-c type chemokine (331). Induction of RANTES usually occurs at a pretranslational level. In T cells, RANTES engagement of the cytokine receptor CCR5 results in Janus kinase kinase and p38 MAPK activation, which in turn leads to downstream target gene activity (332). RANTES has also been implicated in human autoimmunity. Moreover, it has been detected in the thyroid gland of patients with GD (333).
The mechanisms through which chemokines and related molecules alter the migration of immunocompetent cells remain uncertain but probably relate to complex interactions with these cells. The notion that lymphocytes and other target cells might discriminate a concentration gradient across their diameters and adjust their movement toward higher concentrations is probably incorrect. But regardless of the mechanisms involved in their actions, these small molecules are of substantial importance in defining cell movements and infiltration of tissues, such as those targeted in GD.
Human fibroblasts express high levels of IL-16 mRNA under basal conditions but not pro-IL-16 or mature IL-16 protein. In contrast, RANTES mRNA is undetectable in these cells under control conditions. When the fibroblasts are treated with proinflammatory cytokines, such as IL-1, TNF-, TNF-ß, or leukoregulin, they express high levels of mature IL-16 and RANTES proteins that are released from the cell layers. Moreover, both proteins are active and competent to initiate T cell migration (334). Inhibitors of caspase-3 activity block the release of IL-16 from fibroblasts but fail to influence RANTES expression. Glucocorticoids block the expression of both molecules. Greater than 90% of the T cell migratory activity generated by cytokine-treated fibroblasts can be attributed to IL-16 or RANTES, suggesting that these two chemoattractants represent predominant molecular triggers, emanating from fibroblasts that orchestrate lymphocytic infiltration at sites of tissue injury and repair.
If cytokine-activated fibroblasts from many tissues express high levels of IL-16, RANTES, and other important chemoattractant molecules, it is unclear why the manifestations of GD are not more generalized. One possible explanation concerns a global infiltration of immunocompetent cells to many tissues and the differential impact those cells might have on the infiltrated tissues. That model would be consistent with lymphocyte-derived activating molecules exerting site-dependent effects on fibroblasts and other residential cells in a particular anatomic region. From the body of evidence thus far advanced, it would appear that orbital fibroblasts are more susceptible to activation by certain proinflammatory cytokines such as leukoregulin, IL-1, and CD154. Thus, in the context of orbital tissues being infiltrated with lymphocytes and other bone marrow-derived cells, the fibroblasts in residence appear to respond more robustly to cytokines and other factors produced by the immune competent cells than are other types of fibroblasts. Unfortunately, the vast majority of evidence supporting this hypothesis has been generated from cells in culture. Although these findings tend to depict orbital fibroblasts as exhibiting certain behavior, conclusive proof awaits results of studies conducted in vivo in either human beings or animal models.
E. Fibroblasts from the orbit synthesize high levels of hyaluronan
A hallmark feature of the connective tissue remodeling encountered in GO and dermopathy is the accumulation of the linear polymer, hyaluronan (262). The enormous water binding capacity of hyaluronan attracts hydration of connective tissue and accounts at least in part for the expansion of tissues and the expulsion of the eye beyond the normal boundaries of the bony orbit. The fibroblast is an important source of hyaluronan and the other abundant GAGs such as heparin, chondroitin sulfate, and dermatan sulfate (262). Although these cells produce substantial amounts of sulfated GAG under most culture conditions, the role of these complex sugars in the pathogenesis of GO is uncertain. Moreover, the most important impact of cytokine action on orbital fibroblast GAG synthesis relates to changes in hyaluronan production levels (214, 311). Unfortunately, no stringent and quantitative analysis of the GAG content in the orbital connective tissues affected by GO currently exists. Most evidence for disruption of the normal profile of complex carbohydrates in GO rests on rather old reports utilizing nonspecific and highly subjective methodologies. Thus, studies involving assessment of biosynthetic profiles found in cultured orbital fibroblasts provide the best clues concerning the net contribution of this cell type to the changes seen in tissue economy relevant to GO. Extrapolations made to in vivo disease processes might be misleading. Clearly, a careful analysis of GAG content in involved orbital tissues would provide important insights. Moreover, the contribution to net GAG synthesis of other cell types residing in the orbit, such as the cells of the extraocular muscles and the endothelium, need be assessed. In addition, the potential roles for these and other cells, such as those derived from the bone narrow, in degrading GAG needs to be defined because alterations in disposal could account for the accumulation seen in GO.
When orbital fibroblasts are treated with proinflammatory cytokines, they synthesize high levels of hyaluronan. The levels of production are considerably higher than those found in dermal fibroblasts. When orbital fibroblasts are exposed to IFN-, the increase in hyaluronan accumulation is approximately 50%, whereas the effect is absent in dermal cultures (311). Leukoregulin on the other hand increases hyaluronan synthesis by up to 15-fold (214). The induction is dependent on de novo protein synthesis and can be attenuated with dexamethasone. Moreover, hyaluronan production is not influenced when cells are treated with inhibitors of PG synthesis (214). Pulse-chase studies reveal that hyaluronan decay is nil in these cultures, indicating that net synthesis is increased under cytokine-induced conditions.
The mechanisms involved in hyaluronan synthesis have been elucidated recently with the identification and cloning of three members of the HAS family, designated HAS1, 2, and 3 (271, 272, 273). These proteins are thought to catalyze the same rate-limiting and terminal reactions leading to the initiation and chain elongation of mature hyaluronan molecules. In orbital fibroblasts, the most abundant HAS mRNA is that encoding HAS2 (217). Although the levels of the respective mRNAs are quite low in unprovoked fibroblasts, IL-1 and leukoregulin up-regulate all three transcripts severalfold above basal abundance. The effect of IL-1 was transient, with the peak increase in mRNAs occurring 6–12 h after addition of the cytokine into the culture medium. Moreover, the state of cell confluence appeared to influence the magnitude of response to IL-1ß. A substantial induction of HAS2 also occurred after treatment with epidermal growth factor-1 (216). Glucocorticoids, which attenuate cytokine-dependent hyaluronan synthesis in orbital fibroblasts, can block the induction of HAS by IL-1 and leukoregulin. It is of potential importance that human fibroblasts express substantial levels of all three transcripts. The substrate requirements for each enzyme appear to be similar, although the profile of hyaluronan chain lengths from each isoform may differ. Why the same cells should express multiple similar enzymes is uncertain but could relate to their potentially differing roles in development or response to molecular signals. The enzyme immediately upstream from the HAS proteins, UDP-GD, is critical to the synthesis of hyaluronan, chondroitin sulfate, and heparan sulfate. It has recently been cloned and found to be inducible by cytokines in orbital fibroblasts (274). That induction is also time-dependent. Moreover, cycloheximide could induce the transcript after 6 h of treatment, suggesting that the UDP-GD acts as an immediate early gene in orbital fibroblasts. Whether this enzyme might ultimately prove rate-limiting under at least some tissue conditions is not currently known. Moreover, the cell signaling upstream from either HAS or UDP-GD in fibroblasts has not been identified. These cascades might prove important targets for the therapeutic intervention in GO and other conditions in which hyaluronan accumulation becomes disordered.
Another important factor governing the accumulation of ECM components in GO relates to the degradation of GAGs. Orbital fibroblasts do not express hyaluronidase in culture (277), a characteristic of other human fibroblasts (216, 269, 276). When fibroblast monolayers are incubated with medium containing radiolabeled hyaluronan, the molecule remains intact for many days with no evidence of degradation, regardless of the treatment. Whether these cells express hyaluronidases in situ but lose their ability to degrade the GAGs in culture is uncertain. Alternatively, other cells, either residential or those trafficked to the orbit during inflammation or fibrosis, are involved in hyaluronan turnover. The net increase in hyaluronan associated with the pathogenesis of GO is clearly of considerable importance in disrupting the spatial relationships ordinarily maintained between orbital contents.
F. Orbital fibroblasts display high levels of CD40, an important activation molecule
CD40 is a cell-surface glycoprotein receptor first found displayed on B cells (335). It is a member of the TNF- receptor superfamily and has subsequently been identified on other cell types including dendritic, epithelial, and mast cells, and T lymphocytes. CD40 has an important role when displayed on B cells in that lymphocyte activation is initiated through its ligation with CD154, also known as CD40 ligand (336). CD154 is expressed on T cells, mast cells, and some tumor cells. The CD40/CD154 activation bridge is thought to represent a critically important mechanism through which T lymphocytes are believed to activate B cells. More recently, fibroblasts from synovium, lung, and orbital connective tissue have been shown to express high levels of CD40. When CD40 is ligated with CD154, fibroblasts from the lung (337) and orbit (338) express high levels of PGHS-2 and generate extraordinary amounts of PGE2. The induction of PGHS-2 by CD154 is susceptible to glucocorticoids, and these steroids completely abolish the increase in gene expression. Moreover, this induction is mediated through the MAPK pathway in orbital fibroblasts (338). It would appear that the intermediate induction of IL-1 is critical to the up-regulation of PGHS-2. Although IL-1 is induced in a time-dependent manner, IL-1ß is not expressed after CD40 engagement. This selective utilization of IL-1 gene usage in the CD40-activated orbital fibroblast is of substantial mechanistic importance in that IL-1ß can be induced when these cells are treated with IL-1 or leukoregulin. A number of other genes of potential relevance to GO are also up-regulated as a consequence of CD40 ligation in orbital fibroblasts. These include IL-6 and IL-8 (339). IL-6 has been implicated in the pathogenesis of the thyroid glandular components of GD. IL-8 is a C-X-C chemokine that is expressed and released by hematopoietic and nonhematopoietic cells at sites of tissue injury and chronic inflammation. IL-8 also possesses powerful neutrophil and lymphocyte chemoattraction activities. Thus, the selective expression and display of CD40 by fibroblasts from certain tissues and anatomic regions suggests that these cells might respond differently to signals emanating from T cells, and this could represent the basis for anatomic site-selective disease manifestations.
G. Do GD-specific IgGs activate fibroblasts?
Fibroblasts from patients with GD have been examined 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 could enhance collagen synthesis in normal human fibroblasts derived from the skin of the arm. The increase was seen with some but not all monoclonal antibodies generated from human/mouse hybridomas. More recently, Heufelder and Bahn (341) reported that IgGs from patients with GO could increase intercellular adhesion molecule-1 (ICAM-1) expression in orbital fibroblasts from patients with GD but not in cultures derived from donors without known thyroid disease. That report suggested that certain cytokines could also induce ICAM-1 expression in orbital fibroblasts and that the induction of this molecule was accompanied by important functional events related to cell adhesion. The study focused on the IgG and sera from patients with GO. The question of whether patients with GD but without orbital manifestations could also induce ICAM-1 expression was left unexplored. Also absent was an assessment of whether nonorbital fibroblasts from patients with GD could respond to GD-derived IgG. Thus, it is possible that GD-specific IgGs might activate fibroblasts from patients with the disease, regardless of whether they derived from anatomic regions manifesting the disease.
A recent report from Pritchard et al. (342) indicates that sera and IgGs from patients with GD can provoke the expression of T cell chemoattractant activity. When the fibroblasts are exposed to GD-IgG and the resulting conditioned medium is then used to treat target T cells, substantial lymphocyte migration occurs. The vast majority of this activity can be attributed to IL-16 and RANTES. GD-IgG activate the fluoride-resistant acid phosphatase/mammalian target of rapamycin/p70s6 k pathway in fibroblasts from patients with GD (342). In so doing, the expression of IL-16 is dramatically increased. IL-16 mRNA is abundant in untreated human fibroblasts, but the transcript is not translated until the cells are activated (334). The steady-state levels of IL-16 mRNA fail to increase after GD-IgG activation and may decrease several hours later. Specific inhibitors of the FRAP/mTOR/p70s6 k pathway such as rapamycin block the induction of IL-16 in the fibroblasts (342). In contrast, the induction of RANTES appears to be mediated through the activation of another pathway and results in the up-regulation of its mRNA. Rapamycin fails to block the induction of RANTES. Importantly fibroblasts from several anatomic areas of patients with GD are activated to express T cell chemotaxis activity, including areas not usually manifesting clinical disease. Thus, these results suggest that fibroblast activation in GD may be global. Implicit in the findings to date is the possibility that lymphocytes are trafficked to most if not all tissues in GD and that other aspects of the fibroblast phenotype vary between different tissues. Although IL-16 and RANTES have yet to be directly implicated in GD, the finding that both can be selectively induced by disease-specific IgG suggests that such a mechanism might play some role in T 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 bridge elicit responses that differ in magnitude and qualitatively in orbital and nonorbital fibroblasts.
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XIV. Future Perspective |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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The absence of effective and safe therapy for GO is a consequence of poor understanding of the pathogenic mechanisms. It is likely that we will gain better insights through the application of advanced technologies of molecular and cell biology. Clearly, a better definition of T cell/fibroblast interactions should help in the identification of therapeutic targets. The biosynthetic repertoire of orbital fibroblasts appears central to GO. Thus, we might ultimately define ways of specifically targeting hyaluronan and lipid mediator expression in those cells. At an even more fundamental level, characterization of the immunity associated with GD itself might shed needed light on the processes occurring in the orbit. It is likely that the glandular and orbital diseases will ultimately prove to share important common pathogenic mechanisms.
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Footnotes |
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Abbreviations: APC, Antigen-presenting cell; CRP, cross-reactive protein; CTLA4, cytotoxic T lymphocyte antigen-4; DC, dendritic cell; ECM, extracellular matrix; ETSHR, extracellular domain of human TSHR; GAG, glycosaminoglycan; GD, Graves’ disease; GO, Graves’ ophthalmopathy; HAS, hyaluronan synthase; HLA, human leukocyte antigen; ICAM-1, intercellular adhesion molecule-1; IFN-, interferon-; IL-6R, IL-6 receptor; LP, lipoproteins; MBP-ECD, maltose binding protein extracellular domain; MHC, major histocompatibility complex; mPGES, microsomal PGE2 synthase; NF, nuclear factor; PG, prostaglandin; PGHS, PG endoperoxide H synthase; PPAR-, peroxisome proliferator-activated receptor-; RANTES, regulated on activation, normal T cells expressed and secreted; TAO, thyroid-associated ophthalmopathy; TBII, TSH-binding inhibitor Ig; TCR, T cell receptor; TSAb, thyroid-stimulating antibody; TSBAbs, thyroid stimulation blocking antibodies; TSHR, TSH receptor; TSHR-Abs, TSHR-antibodies; UDP, uridine diphosphate; UDP-GD, UDP-glucose dehydrogenase; V, variable; YOP, Yersinia outer membrane protein.
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References |
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TopAbstractI. IntroductionII. Risk Factors for...III. Immunological Basis for...IV. Autoimmune Responses to...V. TSH Receptor as...VI. Evolution of Autoimmune...VII. Graves’...VIII. Genetic and Environmental...IX. Orbital AutoimmunityX. Orbital AutoantigensXI. Role of Orbital...XII. Role of GAGs...XIII. Role of Orbital...XIV. Future PerspectiveReferences |
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 M. Conte, A. Arcaro, D. D'Angelo, A. Gnata, G. Mamone, P. Ferranti, S. Formisano, and F. Gentile A Single Chondroitin 6-Sulfate Oligosaccharide Unit at Ser-2730 of Human Thyroglobulin Enhances Hormone Formation and Limits Proteolytic Accessibility at the Carboxyl Terminus: POTENTIAL INSIGHTS INTO THYROID HOMEOSTASIS AND AUTOIMMUNITY J. Biol. Chem., August 4, 2006; 281(31): 22200 - 22211. [Abstract] [Full Text] [PDF]
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O. F. Lai, N. Zaiden, S. S. Goh, N.-E. Mohamed, L. L. Seah, K. S. Fong, V. Estienne, P. Carayon, S. C. Ho, and D. H C Khoo Detection of thyroid peroxidase mRNA and protein in orbital tissue. Eur. J. Endocrinol., August 1, 2006; 155(2): 213 - 218. [Abstract] [Full Text] [PDF]
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B. Chen, S. Tsui, W. E. Boeglin, R. S. Douglas, A. R. Brash, and T. J. Smith Interleukin-4 Induces 15-Lipoxygenase-1 Expression in Human Orbital Fibroblasts from Patients with Graves Disease: EVIDENCE FOR ANATOMIC SITE-SELECTIVE ACTIONS OF Th2 CYTOKINES J. Biol. Chem., July 7, 2006; 281(27): 18296 - 18306. [Abstract] [Full Text] [PDF]
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D. El Fassi, C. H Nielsen, H. C Hasselbalch, and L. Hegedus The rationale for B lymphocyte depletion in Graves' disease. Monoclonal anti-CD20 antibody therapy as a novel treatment option. Eur. J. Endocrinol., May 1, 2006; 154(5): 623 - 632. [Abstract] [Full Text] [PDF]
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J. A. Gilbert, A. G. Gianoukakis, S. Salehi, J. Moorhead, P. V. Rao, M. Z. Khan, A. M. McGregor, T. J. Smith, and J. P. Banga Monoclonal pathogenic antibodies to the thyroid-stimulating hormone receptor in Graves' disease with potent thyroid-stimulating activity but differential blocking activity activate multiple signaling pathways. J. Immunol., April 15, 2006; 176(8): 5084 - 5092. [Abstract] [Full Text] [PDF]
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 K. J. Land, P. Gudapati, M. H. Kaplan, and G. S. Seetharamaiah Differential Requirement of Signal Transducer and Activator of Transcription-4 (Stat4) and Stat6 in a Thyrotropin Receptor-289-Adenovirus-Induced Model of Graves' Hyperthyroidism Endocrinology, January 1, 2006; 147(1): 111 - 119. [Abstract] [Full Text] [PDF]
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S. E. Feldon, D. J. J. Park, C. W. O'Loughlin, V. T. Nguyen, S. Landskroner-Eiger, D. Chang, T. H. Thatcher, and R. P. Phipps Autologous T-Lymphocytes Stimulate Proliferation of Orbital Fibroblasts Derived from Patients with Graves' Ophthalmopathy Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 3913 - 3921. [Abstract] [Full Text] [PDF]
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 S. M. McLachlan, Y. Nagayama, and B. Rapoport Insight into Graves' Hyperthyroidism from Animal Models Endocr. Rev., October 1, 2005; 26(6): 800 - 832. [Abstract] [Full Text] [PDF]
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G. J. Kahaly, S. Pitz, G. Hommel, and M. Dittmar Randomized, Single Blind Trial of Intravenous versus Oral Steroid Monotherapy in Graves' Orbitopathy J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5234 - 5240. [Abstract] [Full Text] [PDF]
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M. Lantz, T. Vondrichova, H. Parikh, C. Frenander, M. Ridderstrale, P. Asman, M. Aberg, L. Groop, and B. Hallengren Overexpression of Immediate Early Genes in Active Graves' Ophthalmopathy J. Clin. Endocrinol. Metab., August 1, 2005; 90(8): 4784 - 4791. [Abstract] [Full Text] [PDF]
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 P. N. Cunningham and R. J. Quigg Contrasting Roles of Complement Activation and Its Regulation in Membranous Nephropathy J. Am. Soc. Nephrol., May 1, 2005; 16(5): 1214 - 1222. [Abstract] [Full Text] [PDF]
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R. Han and T. J. Smith Induction by IL-1{beta} of Tissue Inhibitor of Metalloproteinase-1 in Human Orbital Fibroblasts: Modulation of Gene Promoter Activity by IL-4 and IFN-{gamma} J. Immunol., March 1, 2005; 174(5): 3072 - 3079. [Abstract] [Full Text] [PDF]
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J. L. Wemeau, P. Caron, A. Beckers, V. Rohmer, J. Orgiazzi, F. Borson-Chazot, M. Nocaudie, P. Perimenis, S. Bisot-Locard, I. Bourdeix, et al. Octreotide (Long-Acting Release Formulation) Treatment in Patients with Graves' Orbitopathy: Clinical Results of a Four-Month, Randomized, Placebo-Controlled, Double-Blind Study J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 841 - 848. [Abstract] [Full Text] [PDF]
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G. Baker, G. Mazziotti, C. von Ruhland, and M. Ludgate Reevaluating Thyrotropin Receptor-Induced Mouse Models of Graves' Disease and Ophthalmopathy Endocrinology, February 1, 2005; 146(2): 835 - 844. [Abstract] [Full Text] [PDF]
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 S. Costagliola, M. Bonomi, N. G. Morgenthaler, J. Van Durme, V. Panneels, S. Refetoff, and G. Vassart Delineation of the Discontinuous-Conformational Epitope of a Monoclonal Antibody Displaying Full in Vitro and in Vivo Thyrotropin Activity Mol. Endocrinol., December 1, 2004; 18(12): 3020 - 3034. [Abstract] [Full Text] [PDF]
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C.-R. Chen, H. Aliesky, P. N. Pichurin, Y. Nagayama, S. M. McLachlan, and B. Rapoport Susceptibility Rather than Resistance to Hyperthyroidism Is Dominant in a Thyrotropin Receptor Adenovirus-Induced Animal Model of Graves' Disease as Revealed by BALB/c-C57BL/6 Hybrid Mice Endocrinology, November 1, 2004; 145(11): 4927 - 4933. [Abstract] [Full Text] [PDF]
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J. Pritchard, S. Tsui, N. Horst, W. W. Cruikshank, and T. J. Smith Synovial Fibroblasts from Patients with Rheumatoid Arthritis, Like Fibroblasts from Graves' Disease, Express High Levels of IL-16 When Treated with Igs against Insulin-Like Growth Factor-1 Receptor J. Immunol., September 1, 2004; 173(5): 3564 - 3569. [Abstract] [Full Text] [PDF]
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T. Cawood, P. Moriarty, and D. O'Shea Recent developments in thyroid eye disease BMJ, August 14, 2004; 329(7462): 385 - 390. [Full Text] [PDF]
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