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Analysis of extraocular muscle‐infiltrating T cells in thyroid‐associated ophthalmopathy (TAO)
Abstract
TAO is characterized by an autoimmune process affecting the orbital contents. T cells have been suggested to have a major role in pathogenesis, but so far only limited data are available to clarify the extraocular muscle (EOM)-infiltrating T cell phenotype, antigenic reactivity and cytokine profile in TAO patients. In the present study, biopsies of affected EOM were taken and the infiltrating T cells isolated and expanded in vitro with mitogen. Their phenotype was determined by flow cytometric (FACS) analysis and compared with peripheral blood-derived T cell lines, treated in the same way from the same patient. Cytokines present in the supernatant after mitogen stimulation of the T cell lines were assayed by ELISA. In addition, cytokine mRNA present at the time of biopsy was determined by rapid RNA extraction from EOM and reverse transcription-amplification with specific cytokine oligonucleotide probes (IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL- 15, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha)). In the T cell lines from two patients, proliferation assays were carried out with antigens derived from thyroid gland, EOM and a thyrotropin (TSH) receptor preparation. Most T cell lines were CD4+, CD45RO+, and TCR alpha/beta+, both from the EOM and the peripheral blood. A wide variety of cytokines was detected by analysis of supernatants or mRNA, but the profiles were not identical comparing the two approaches. However, IL-4 was detected by both. Dose-dependent proliferation was observed in response to thyroid extract in a biopsy-derived T cell line. In conclusion, EOM-infiltrating T cells from patients with TAO, expanded in vitro, were chiefly CD4+ and produced a mixture of cytokines, including IL-4. The proliferation data suggest that there are thyroid-reactive T cells in EOM.
... The same status was found in the four corresponding PBMC samples (three were mostly CD4 + cells (89%-98%)) of each patient. They further reported detectable T cell receptor (TCR) gene expression in 10 out of 12 EOMs collected from the other five patients and in all five EOMs collected from three control subjects (41). The discrepancy of CD4 + and CD8 + T cell subsets in the above findings may lie in the small number of patients, the heterogeneity of patients involved in the different studies, and the different research methods. ...
... Expression of Ifng, Il13, Il1b, and Il12p40 was not detected in these EOMs. Il6 and Il8 were the only cytokine genes expressed in two out of five EOMs from three control subjects (41). It should be considered that gene and protein expressions are not complete coincident. ...
Graves’ orbitopathy (GO), also known as thyroid-associated ophthalmopathy, is the most common ocular abnormality of Graves’ disease. It is a disfiguring, invalidating, and potentially blinding orbital disease mediated by an interlocking and complicated immune network. Self-reactive T cells directly against thyroid-stimulating hormone receptor-bearing orbital fibroblasts contribute to autoimmune inflammation and tissue remodeling in GO orbital connective tissues. To date, T helper (Th) 1 (cytotoxic leaning) and Th2 (antibody leaning) cell subsets and an emerging role of Th17 (fibrotic leaning) cells have been implicated in GO pathogenesis. The potential feedback loops between orbital native residential CD34⁻ fibroblasts, CD34⁺ infiltrating fibrocytes, and effector T cells may affect the T cell subset bias and the skewed pattern of cytokine production in the orbit, thereby determining the outcomes of GO autoimmune reactions. Characterization of the T cell subsets that drive GO and the cytokines they express may significantly advance our understanding of orbital autoimmunity and the development of promising therapeutic strategies against pathological T cells.
... [13][14][15] The majority of the infiltrating MNCs were identified as T cells, predominantly CD8 + T cells in the connective tissue, 15 and activated CD4 + T cells in the extraocular muscles. 16 The nature and significance of the autoimmune reactions within the thyroid that lead to the development of ophthalmopathy have recently been addressed in our laboratory. In a previous study (Al-Ansari, Wall et al., unpublished observations), we have found that variation in the difference between thyroidal and peripheral blood CD8 + T cells and CD8 + regulatory T (T-reg) cells could be relevant to the development of ophthalmopathy in patients with GD. ...
... 13,14,19,20 The majority of the cell infiltrates in the orbit of GO are T cells with a predominance of CD8 + T cells in the connective tissue [13][14][15] and CD4 + T cells in the extraocular muscles. 16 Other cell infiltrates include B cells, macrophages and mast cells. [13][14][15] Despite not being considered as a major player in autoimmunity in previous decades, recent evidence has shown major destructive as well as protective roles for CD8 + T cells in autoimmunity. ...
Purpose/aim of the study: Graves’ ophthalmopathy (GO) is closely related to the thyroid autoimmune disorder Graves’ disease. Previous studies have suggested roles for thyroidal CD8⁺ T cells and autoimmunity against calsequestrin-1 (CASQ)-1 in the link between thyroidal and orbital autoimmune reactions in GO. A role for autoimmunity against CollXIII has also been suggested. In this study, we aimed to investigate correlations between some thyroidal and peripheral blood T-cell subsets and thyroidal T-cell reactivity against CASQ1 and CollXIII in patients with GO.
Materials and methods: Fresh thyroid tissues were processed by enzyme digestion and density gradient to isolate mononuclear cells (MNCs). Peripheral blood MNCs were also isolated using density gradient. Flow-cytometric analysis was used to identify the various T-cell subsets. T -cell reactivity to CASQ1 and CollXIII was measured by a 5-day culture of the MNCs and BrdU uptake method.
Results: We found a positive correlation between thyroidal CD8⁺ T cells and CD8⁺ T-regulatory (T-reg) cells in patients with GO. Thyroidal T cells from two out of the three patients with GO tested (66.7%) showed a positive response to CASQ1, while thyroidal T cells from none of the six Graves’ Disease patients without ophthalmopathy (GD) tested showed a positive response to this antigen. Thyroidal T cells from these patient groups however, showed no significant differences in their response to CollXIII.
Conclusions: Our observations provide further evidence for a possible role of thyroidal CD8⁺ T cells, CD8⁺ T-reg cells and the autoantigen CASQ1 in the link between thyroidal and orbital autoimmune reactions of GO.
... A previous study on extraocular muscles revealed infiltration of lymphocytes and plasma cells and deposition of glycosaminoglycans [21]. The lymphocytes were mainly CD4-positive T lymphocytes, which have been reported to produce various cytokines, such as interleukin-4 [22]. Therefore, the present study focused on the factors associated with extraocular muscle enlargement in patients with TED. ...
Purpose
This study aimed to investigate the factors affecting extraocular muscle enlargement in thyroid eye disease (TED).
Study design
Retrospective study.
Methods
The thyroid-stimulating hormone (TSH) receptor antibody (TRAb), thyroid-stimulating antibody (TSAb), antithyroid peroxidase antibody (ATPO), and antithyroglobulin antibody (ATG) levels in patients diagnosed with TED who underwent orbital magnetic resonance imaging were assessed. The control group comprised the contralateral eye of patients who underwent orbital magnetic resonance imaging (MRI) for unilateral eyelid tumors or orbital disease. The thickness of the bilateral rectus muscles and superior oblique muscles was measured on orbital MRI. Muscle enlargement was classified as unilateral/bilateral and symmetric/asymmetric. The effects of age, sex, smoking history, TSH, thyroid hormone, and thyroid autoantibodies on the muscle thickness and number of enlarged muscles were assessed by use of simple and multiple regression analyses.
Results
The TED and control groups comprised 41 and 44 cases, respectively. The positivity rate of TSAb in patients with TED was 92.7% higher than that of the other autoantibodies. Muscle enlargement was observed in 29 of the 41 cases (70.7%). Older age and higher TSAb levels were identified as significant factors affecting the total muscle thickness and number of enlarged muscles. Bilateral muscle enlargement and asymmetrical muscle enlargement were observed in 17 (58.6%) and 23 (79.3%) of the 29 cases, respectively. The TSAb levels and age had no significant effect on the type of muscle enlargement.
Conclusions
TSAb showed significant associations with extraocular muscle enlargement. Measurement of TSAb, rather than of TRAb, may be more useful for diagnosing extraocular muscle enlargement in patients with TED.
... Naïve T cells differentiate into T helper (Th) or cytotoxic T cells (Tc), with CD4 + cells supporting humoral immunity and CD8 + cells regulating cell-mediated immune responses [35][36][37]. In TED, there is a notable increase in the CD4/CD8 ratio, potentially indicating a decline in CD8 + T cells [33,38,39]. ...
... In GO patients, mononuclear cell infiltration (CD4 and CD8 T lymphocytes, B lymphocytes, plasma cells, and macrophages) has been shown in the orbital fat, extraocular muscles, and lacrimal gland. TNF, IL-2, IFN-γ, and other inflammatory cytokines released by these cells are particularly potent in the active phase, whereas cytokines such as IL-1, IL-6, and TGF-β are released by fibroblasts and adipocytes in the later stages (9,10). In addition to the effects of GO on the orbital tissues, there are intraocular complications such as increased IOP, choroidal folds, and optic disc edema (11). ...
Objectives
The aim of this study was to assess intraocular inflammation in patients with active and inactive Graves’ ophthalmopathy (GO) using an aqueous laser flash meter and to assess its relationship with thyroid hormones, antibodies, and clinical activity score (CAS).
Methods
Forty patients (29 females and 11 males) were included in the study. The patients were divided into two groups according to CAS; patients with CAS <3 (inactive) were included in Group 1 and patients with CAS ≥3 (active) were included in Group 2. The laser flare meter was used to measure the flare of aqueous humor. Each patient’s ocular findings, thyroid hormone, and antibody levels were also recorded.
Results
The mean age of patients was 46.88±11.79 years in Group 1 and 44.50±12.59 years in Group 2 (p=0.555). The mean CAS was 0.88±0.65 in Group 1 and 3.57±0.85 in Group 2 (p<0.001). The mean aqueous flare was 6.5±2.2 ph/ms in Group 1 and 7.0±6.4 ph/ms in Group 2 (p=0.73). Hertel exophthalmometry, intraocular pressure (IOP), antithyroglobulin antibody, and thyroid stimulating hormone receptor antibody (TRAb) levels were similar in both groups (each p>0.05). There was no correlation between aqueous flare value and CAS, Hertel exophthalmometry, IOP, thyroid hormone, and antibody levels (each p>0.05). There was a significant correlation between CAS and antibody levels (each p<0.05).
Conclusion
Flare values that are not much above the normal range may be an indication that intraocular inflammation is not elevated in GO patients. This suggests that the damage to the blood-aqueous barrier in these patients is not severe enough to increase intraocular inflammation.
... The recruitment and infiltration of various immune cells into the orbit determine pathological changes in orbital tissues [20]. These cells are primary CD4+ T cells, and there are also minor populations of CD8+ cells, macrophages, plasma cells, and B cells, which focally and diffusely infiltrate the adipose tissues, extraocular and levator muscles, and lacrimal glands in active TAO [21][22][23]. Cytokines from immune cells led to the production of ongoing inflammation in the orbital adipose tissue and fibrous tissue of extraocular muscles, which caused increased pressure within the bony cavity [24,25]. ...
Thyroid-associated ophthalmopathy (TAO), also known as thyroid eye disease (TED) or Graves’ orbitopathy (GO), is a complex autoimmune condition causing visual impairment, disfigurement, and harm to patients’ physical and mental health. The pathogenesis of TAO has not been fully elucidated, and the mainstream view is that coantigens shared by the thyroid and orbit trigger remodeling of extraocular muscles and orbital connective tissues through an inflammatory response. In recent years, cytokines and the immune responses they mediate have been crucial in disease progression, and currently, common evidence has shown that drugs targeting cytokines, such as tocilizumab, infliximab, and adalimumab, may be novel targets for therapy. In this review, we summarize the research development of different cytokines in TAO pathogenesis in the hope of discovering new therapeutic targets.
... Inflammation in the orbit of patients with GO is accepted to be caused by autoimmune responses and inflammatory cytokine release [17,18]. RNA methylation has been reported to regulate T-cell homeostasis and the inflammatory response by targeting inflammatory pathways [19]. ...
Purpose
To investigate the role of N6-methyladenosine (m ⁶ A) RNA modification in the pathogenesis of Graves' ophthalmopathy (GO).
Methods
Surgically excised extraocular muscles from 7 patients with GO and 5 subjects without GO were used. The global m ⁶ A levels in the specimens were determined using an m ⁶ A RNA methylation quantification kit. RNA sequencing (RNA-seq) was used to analyze the molecules involved in the regulation of m ⁶ A RNA methylation and the differential expression of mRNAs between the two groups (4 eyes, respectively). The expression of m ⁶ A RNA modification genes was evaluated by real-time PCR. The functional implications of the gene alterations between the GO and control specimens were determined by Gene Ontology analysis.
Results
The m ⁶ A level was significantly increased in the specimens of GO patients compared to the control specimens ( P < 0.05). The expression of m ⁶ A methylation regulators, such as WT1 associated protein (WTAP), alkylation repair homolog protein 5 (ALKBH5), E74 like ETS transcription factor 3 (ELF3), YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), YTHDF3 and YTH domain containing 2 (YTHDC2), was significantly upregulated ( P < 0.05). Gene Ontology enrichment analysis showed that the most highly upregulated genes and biological pathways were related to the immune response and inflammatory processes such as lymphocyte activation, leukocyte differentiation, cytokine production and cytokine-mediated signaling pathways.
Conclusions
Our results suggest that m ⁶ A methylation may play a critical role in the pathogenesis of GO and that targeting genes that regulate m ⁶ A methylation may provide a new therapeutic approach for GO.
... [35] T cells infiltrates in GO orbital tissues are predominantly CD4 + , with some studies suggesting presence of both CD4 + and CD8 + T cells. [36][37][38][39] A study [20] we included, also suggested that the imbalance of T cell subsets may be an important factor leading to GO by detecting peripheral blood T cell subsets before and after treatment. ...
Background:
Graves ophthalmopathy (GO) is one of the remaining enigmas in thyroidology. Glucocorticoids (GCs) are strongly recommended but their effects are not completely satisfactory and adverse reactions can occur. Tripterygium glycosides (TG) is a promising component extracted from Tripterygium wilfordii Hook F (TwHF), and numerous patients with GO have benefited from it. However, its practical application value is still unclear. The aim of this systematic review and meta-analysis was to investigate the efficacy and safety of TG for patients with GO.
Methods:
By retrieving the PubMed, Embase, the Cochrane Library, CNKI, VIP, CBM, and WanFang Databases, the open published randomized controlled trials (RCTs) related to TG in the treatment of GO were collected. And inclusion and exclusion criteria were established. The Cochrane bias risk assessment tool conducts the evaluation of included studies, and meta-analysis was performed using Revman 5.3 software.
Trial registration number:
PROSPERO CRD42019131915.
Results:
A total of 19 trials (involving 1517 GO patients) were included in this review with generally acceptable validity of included RCTs. TG therapy brought about a significantly higher efficacy rate compared with non-TG treatments (RR: 1.40; 95% CI: 1.31-1.49). Subgroup meta-analysis showed that TG with or without immunosuppressive therapies were all better than controls: with GC (RR: 1.36; 95% CI: 1.27-1.46), with multiple intensification of immunosuppressive therapies (RR: 1.91; 95% CI: 1.37-2.67), with no immunosuppressive therapies (RR: 1.39; 95% CI:1.21-1.59); the dosage of TG for 15-60 mg/d (RR: 1.41; 95% CI: 1.30-1.53) were better compared with for ≥90 mg/d (RR: 1.47; 95% CI: 1.29-1.68); the course of treatment for ≤3 months (RR: 1.43; 95% CI: 1.33-1.52) was better than controls, but when >3 months (RR: 1.15; 95% CI: 0.94-1.41) there was no significant differences. After treatment, the degree of exophthalmus (SMD: -2.55; 95% CI: -2.93 to 2.17), the recurrence rate of 1 year (RR: 0.45; 95% CI: 0.27-0.74), and adverse reactions rate (RR: 0.32; 95% CI: 0.20-0.53) were all lower, while the CAS was no obvious gap in 2 groups (SMD: 0.08; 95% CI: -0.60 to 0.75).
Conclusions:
This review found that TG has some advantages in treating GO, especially in improving clinical efficacy and reducing adverse reactions. Nevertheless, large sample, multi-center, reasonable design, and high quality clinical studies are still needed for further verification.
... T-cell infiltrates in GO orbital tissues are predominantly CD4 + , with some studies suggesting the presence of both CD8 + and CD4 + T cells [9][10][11][12]. Th1-like cytokine profile is expressed mainly in GO retrobulbar tissue [10][11][12][13]. Th1like cytokine expression profile consisting of interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-1β, and IL-6 occurs mainly in GO extraocular muscles, whereas IL-4 and IL-10, Th2-type cytokines, are expressed in orbital fat [14]. ...
Graves’ ophthalmopathy (GO) is an inflammatory autoimmune disorder of the orbital adipose tissue and extraocular muscles, and it is associated with Graves’ disease (GD). GO is triggered by binding and activation of orbital fibroblasts by autoantibodies (TSI) direct against thyroid-stimulating hormone receptor (TSHR) and insulin-like growth factor 1 (IGF-1R), which is highly expressed within the orbit. Moreover, interaction of T cells with orbital fibroblasts that involve T-cell receptor (TCR), autoantigen, and major histocompatibility complex class II (MHC II) molecule, as well as CD40:CD154 signalling, activates p38, ERK 1/2, and JNK pathways. These processes induce fibroblast activation, proliferation, and secretion of chemokines and inflammatory cytokines to maintain inflammation within the orbit. Furthermore, increased hyaluronic acid production and fibroblast differentiation into adipocytes and myofibroblasts leads to development of GO. The elevated number of molecular factors such as PDGF, IL1-β, IL-4, IL-6, IL10, IL-8, IL-16, IL-33, HGF, ICAM-1, osteopontin, CTLA-4, and TGF-β are discussed in the paper. Some of them are key markers of disease stage. Better understanding of GO pathogenesis leads to development of new therapeutic options.
... T cell infiltrates in TED orbital tissues are predominantly CD4+, with some studies suggesting presence of both CD8+ and CD4+ T cells. Th1-like cytokine profile predominates in TO retrobulbar tissue [11][12][13][14]. ...
Recent new insights into the molecular basis of thyroid eye disease have led to the use of more specific therapies such as monoclonal antibodies This review explores the traditional immunosuppressant therapy for TED, highlighting the basis for emergent recent medications, possible treatment options and, eventually possible new general recommendation for management of TED. Data has been retrieved from the literature searching on Pubmed. Steroid therapy remains the first line therapy for moderate/severe and severe vision threatening TED The use of some traditional nonspecific immunosuppressant such as mycophenolate, cyclosporine and azathioprine seems useful in combination with steroid therapy to achieve stable results in the long term; methotrexate is useful as steroid-sparing medications and in steroid resistant or intolerant patients. In recent years, many scientific reports have showed the effectiveness of biological immunosuppressive agents in the management of TED. Etanercept, adalimumab, and tocilizumab have shown to be effective in reduction of the inflammatory signs with the possible advantage to prevent relapse of the disease. Particularly Tociliuzumab seems very effective as second line therapy, after steroid failure. Teprotumumab may control the disease activity and it seems to be very effective in preventing severity disease progression. Infliximab might be useful in severe TED with optic nerve compression resistant to steroid and decompression. Indeed, the actual incidence of adverse effects is not well assessed yet, therefore the use should be limited at those cases that really need an alternative therapy to steroid, handled by an expert multidisciplinary team.
... Orbital fibroblasts are both the major targets of inflammatory cytokines released by infiltrating immune cells as well as active participants in pathological progression (Bahn, 2010(Bahn, , 2015(Bahn, , 2016van Steensel et al., 2012a,b;Feng et al., 2017;reviewed in Bahn, 2015;Dik et al., 2016). In early GO, diffuse infiltration of primarily CD4+ T cells are the predominant microenvironmental effectors in the orbit but CD8+ T cells, macrophages, plasma cells and B cells are also evident in the EOM and adipose tissue (Pappa et al., 1997;Eckstein et al., 2004). Type 1 helper T cells produce inflammatory cytokines including IL-2, interferon-γ and TNFα the initial stages (Bahn, 2010). ...
Graves’ ophthalmopathy (GO), a complication of Graves’ disease (GD), is typified by orbital inflammation, ocular tissue expansion and remodeling and, ultimately, fibrosis. Orbital fibroblasts are key effectors of GO pathogenesis exhibiting exaggerated inflammatory and fibroproliferative responses to cytokines released by infiltrating immune cells. Activated orbital fibroblasts also produce inflammatory mediators that contribute to disease progression, facilitate the orbital trafficking of monocytes and macrophages, promote differentiation of matrix-producing myofibroblasts and stimulate accumulation of a hyaluronan-rich stroma, which leads to orbital tissue edema and fibrosis. Proteomic and transcriptome profiling of the genomic response of ocular and non-ocular fibroblasts to INF-γ and TGF-β1 focused on identification of translationally-relevant therapeutic candidates. Induction of plasminogen activator inhibitor-1 (PAI-1, SERPINE1), a clade E member of the serine protease inhibitor (SERPIN) gene family and a prominent regulator of the pericellular proteolytic microenvironment, was one of the most highly up-regulated proteins in INF-γ- or TGF-β1-stimulated GO fibroblasts as well as in severe active GD compared to patients without thyroid disease. PAI-1 has multifunctional roles in inflammatory and fibrotic processes that impact tissue remodeling, immune cell trafficking and survival as well as signaling through several receptor systems. This review focuses on the pathophysiology of the GO fibroblast and possible targets for effective drug therapy.
... At the opposite, IL-10, IL-13 and IL-6 are produced by Th2 cells. The involvement of both CD4+ T cell populations is described in TAO disease [53][54][55][56] . Their predominance seems tissue-specific: Th1 cells are more detected in extracellular muscles whereas Th2 cells are located preferably in orbital tissues. ...
The aim was to investigate the levels of cytokines and soluble IL-6R in the tears of patients with thyroid-associated orbitopathy (TAO) disease. Schirmer's test was adopted to collect tears from TAO patients (N = 20, 17 women, mean age (±SD): 46.0 years (±13.4)) and healthy subjects (N = 18, 10 women, 45.4 years (±18.7)). Lacrimal cytokines and soluble IL-6R (sIL-6R) were measured using a 10-plex panel (Meso Scale Discovery Company) and Invitrogen Human sIL-6R Elisa kit, respectively. Tear levels of IL-10, IL-12p70, IL-13, IL-6 and TNF-α appeared significantly higher in TAO patients than in healthy subjects. Interestingly, IL-10, IL-12p70 and IL-8 levels increased in tears whatever the form of TAO whereas IL-13, IL-6 and TNF-α levels were significantly elevated in inflammatory TAO patients, meaning with a clinical score activity (CAS) ≥ 3, compared to controls. Furthermore, only 3 cytokines were strongly positively correlated with CAS (IL-13 Spearman coeff. r: 0.703, p = 0.0005; IL-6 r: 0.553, p = 0.011; IL-8 r: 0.618, p = 0.004, respectively). Finally, tobacco use disturbed the levels of several cytokines, especially in patient suffering of TAO. The differential profile of lacrimal cytokines could be useful for the diagnosis of TAO patients. Nevertheless, the tobacco use of these patients should be taken into account in the interpretation of the cytokine levels.
... GO is caused by inflammation in the orbital tissues and fibroblast proliferation secondary to abnormal cellular autoimmune responses characterized by CD4+ and CD8+ T cell infiltration and expansion (5,6) and excessive production of IL-1, IL-6, RANTES, IL-8, interferon-γ and TNF-α, which promote fibroblast proliferation and differentiation (7). There are phenotypic and functional differences between fibroblasts of orbital muscle and adipose tissues (8,9). ...
Objective:
This study sought to compare patients with thyroid eye disease (TED) and normal controls with respect to the expression of the GRα, IKK1, IκB, c-Fos (AP-1), NF-kB, and 11β-HSD1 genes in orbital fat (OF) and extraocular muscle (EOM).
Design and methods:
A prospective study design was used to evaluate 34 TED patients and 38 healthy controls. OF was harvested from 33 TED patients and 27 controls. EOM biopsies were obtained from 32 TED patients and 18 controls. Samples were examined by real-time PCR and evaluated using appropriate statistical analyses with a significance cut-off of p<0.05.
Results:
GRα mRNA levels were higher in TED EOM [median 213 (96-376)] than in control EOM [78 (34-138)] (p<0.001), and NF-kB expression was elevated in TED muscle [223 (31-520)] relative to that in control muscle [8 (6-31)] (p<0.001). 11β-HSD1 expression was higher in TED EOM [0.78 (0.47-2.01)] than in control EOM [0.22 (0.09-0.51)] (p<0.001). Levels of IKK1, IκB, and c-Fos were higher in TED EOM [115 (20-223), 111 (54-299), and 0.11 (0.03-0.19), respectively] than in control EOM [5.8 (2-13), 21 (5-52), and 0.05 (0.001-0.03), respectively] (p<0.001).
Conclusion:
Tissues involved in GO exhibited different mRNA levels of GRα, IKK1, IκB, c-Fos (AP-1), NF-kB, and 11β-HSD1. Gene expression in OF was similar for TED patients and controls. IKK1, IκB, c-Fos, NF-kB, and 11β-HSD1 mRNA levels were higher in TED EOM than in control EOM. NF-kB was disproportionally elevated compared with GRα; this finding was indicative of a local proinflammatory profile.
... T cell infiltrates in TO orbital tissues are predominantly CD4+, with some studies suggesting presence of both CD8+ and CD4+ T cells. [45][46][47][48] Th1-like cytokine profile predominates in TO retrobulbar tissue. 45 48 Th1-like cytokine expression profile consisting of interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-1β and IL-6 has been detected mainly in TO extraocular muscles, whereas IL-4 and IL-10, Th2-type cytokines were detected predominantly in orbital fat. ...
Orbital changes in thyroid orbitopathy (TO) result from de novo adipogenesis, hyaluronan synthesis, interstitial oedema and enlargement of extraocular muscles. Cellular immunity, with predominantly CD4+ T cells expressing Th1 cytokines, and overexpression of macrophage-derived cytokines, perpetuate orbital inflammation. Orbital fibroblasts appear to be the major effector cells. Orbital fibroblasts express both thyrotropin receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R) at higher levels than normal fibroblasts. TSHR expression increases in adipogenesis; TSHR agonism enhances hyaluronan production. IGF-1R stimulation leads to adipogenesis, hyaluronan synthesis and production of the chemokines, interleukin (IL)-16 and Regulated on Activation, Normal T Cell Expression and Secreted, which facilitate lymphocyte trafficking into the orbit. Immune activation uses a specific CD40:CD154 molecular bridge to activate orbital fibroblasts, which secrete pro-inflammatory cytokines including IL-1β, IL-1α, IL-6, IL-8, macrophage chemoattractant protein-1 and transforming growth factor-β, to perpetuate orbital inflammation. Molecular pathways including adenylyl cyclase/cyclic adenosine monophosphate, phophoinositide 3 kinase/AKT/mammalian target of rapamycin, mitogen-activated protein kinase are involved in TO. The emergence of a TO animal model and a new generation of TSHR antibody assays increasingly point towards TSHR as the primary autoantigen for extrathyroidal orbital involvement. Oxidative stress in TO resulting from imbalances of the oxidation-reduction state provides a framework of understanding for smoking prevention, achieving euthyroidism and the use of antioxidants such as selenium. Progress has been made in the understanding of the pathogenesis of TO, which should advance development of novel therapies targeting cellular immunity, specifically the CD40:CD40 ligand interaction, antibody-producing B cells, cytokines, TSHR and IGF-1R and its signalling pathways. Further studies in signalling networks and molecular triggers leading to burnout of TO will further our understanding of TO.
... The autoimmune nature of GO is evidenced by the presence within the orbit of a perivascular and diffuse infiltration with mononuclear cells, including predominately CD4 + T cells with occasional populations of CD8 + cells, B cells, and macrophages [1,23]. CD4 + T cells were primarily γδT cells, while αβT helper cells were rare [24]. ...
Environmental, genetic, and immune factors are at play in the development of the variable clinical manifestations of Graves' ophthalmopathy (GO). Among the environmental contributions, smoking is the risk factor most consistently linked to the development or worsening of the disease. The close temporal relationship between the diagnoses of Graves' hyperthyroidism and GO have long suggested that these 2 autoimmune conditions may share pathophysiologic features. The finding that the thyrotropin receptor (TSHR) is expressed in orbital fibroblasts, the target cells in GO, supported the notion of a common autoantigen. Both cellular and humeral immunity directed against TSHR expressed on orbital fibroblasts likely initiate the disease process. Activation of helper T cells recognizing TSHR peptides and ligation of TSHR by TRAb lead to the secretion of inflammatory cytokines and chemokines, and enhanced hyaluronic acid (HA) production and adipogenesis. The resulting connective tissue remodeling results in varying degrees extraocular muscle enlargement and orbital fat expansion. A subset of orbital fibroblasts express CD34, are bone-marrow derived, and circulate as fibrocytes that infiltrate connective tissues at sites of injury or inflammation. As these express high levels of TSHR and are capable of producing copious cytokines and chemokines, they may represent an orbital fibroblast population that plays a central role in GO development. In addition to TSHR, orbital fibroblasts from patients with GO express high levels of IGF-1R. Recent studies suggest that these receptors engage in cross-talk induced by TSHR ligation to synergistically enhance TSHR signaling, HA production, and the secretion of inflammatory mediators.
© Georg Thieme Verlag KG Stuttgart · New York.
... The pathophysiology of GO comprises various cell types, inflammatory mediators and autoantibodies that interact with each other, resulting in orbital inflammation and tissue expansion. Immune cells involved in GO include at least T-lymphocytes, B-lymphocytes, monocytes, macrophages and mast cells, which activate orbital fibroblasts either via secreted factors or direct cell-cell contact [9,10] . Orbital fibroblast activation with resultant production of cytokines and glycosaminoglycans (especially hyaluronan), proliferation and adipocyte differentiation is considered to fulfill a central role in orbital tissue inflammation and expansion in GO ( fig. 1 ). ...
Activation of orbital fibroblasts resulting in excessive proliferation, cytokine and hyaluronan production and differentiation into adipocytes, is a main determinant of orbital tissue inflammation and tissue expansion in Graves' ophthalmopathy (GO). During the last years we have shown that the platelet-derived growth factor (PDGF) isoforms PDGF-AA, PDGF-AB and PDGF-BB are increased in orbital tissue from GO patients with active and inactive disease. These PDGF isoforms exhibit the capacity to stimulate proliferation, hyaluronan and cytokine/chemokine production by orbital fibroblasts. Moreover, PDGF-AB and PDGF-BB increase thyroid stimulating hormone receptor (TSHR) expression by orbital fibroblasts, which enhances the orbital fibroblast activating capacity of the THSR stimulatory autoantibodies present in Graves' disease (GD) patients. Of these PDGF isoforms PDGF-BB exhibits the strongest orbital fibroblast activating effects, which is likely related to its ability to bind both the PDGF-receptor (PDGF-R)α and PDGF-Rβ chains. Thus the PDGF-system fulfills important roles in orbital fibroblast activation in both active and inactive GO, which supports a therapeutic rationale for blocking PDGF signaling in GO. Tyrosine kinase inhibitors (TKIs) may be candidates to target PDGF signaling. Of several TKIs tested dasatinib exhibited the highest potency to block PDGF-R signaling in orbital fibroblasts and may represent a promising compound for the treatment of GO as it was effective at low dosage and is associated with less side effects compared to imatinib mesylate and nilotinib. In this review the contribution of PDGF to the pathophysiology of GO as well as therapeutic approaches to target this PDGF-system will be addressed.
... Focal and diffuse mononuclear-cell infiltration occurs within the extraocular and levator muscles, lacrimal glands, and orbital adipose tissues. These cells are primarily CD4+ T cells, but also CD8+ cells, B cells, plasma cells, and macrophages and type 1 helper T cells 29,30 , producing the cytokines interleukin-2, interferon-γ, and tumor necrosis factor 31 , indicating cell-mediated immunity within the orbit. In later stages of the disease, type 2 helper T cells produce interleukin-4, interleukin-5, and interleukin-10. ...
PurposeThe study was conducted to analyze aqueous flare and its correlations in patients with Graves' ophthalmopathy (GO) undergoing orbital decompression, extraocular muscle, and eyelid surgery. Prospective interventional case series.Patients and Methods
Forty-eight eyes of 27 patients (20 female and 7 male, aged 54.4±5.7) undergoing surgical treatment for GO. Eighteen eyes of nine patients (aged 55.3±3.6) undergoing orbital decompression. Nineteen eyes of 11 patients (aged 54.7±5.6) undergoing extraocular muscle surgery and 13 eyes of 7 patients (aged 53.9±4.9) undergoing eyelid surgery and control group (34 patients aged 53.9±5.1). Laser flare analysis and clinical assessment were performed before surgery and at 1 day, 7 days, and 3 months following surgery.ResultsAqueous flare was significantly higher in patients with GO (14.03±8.45) before intervention than in the control group (7.89±3.56) (P<0.001), and correlated with Clinical Activity Score and intraocular pressure. In the patients undergoing orbital decompression, flare increased from 17.77±10.63 pc/ms to 38.32±13.56 pc/ms on the first day and 41.31±17.19 pc/ms on the seventh day and returned to 16.01±8.58 pc/ms in 3 months. In patients undergoing extraocular muscle surgery flare increased from 13.05±6.50 to 23.04±11.53 pc/ms (P<0.001) on the first day and returned to 18.02±14.09 pc/ms on the seventh day. Eyelid surgery did not change flare values.Conclusions
Orbital decompression disrupts blood-aqueous barrier (BAB). The integrity of BAB returns to preoperative status within 3 months. Extraocular muscle surgery mildly affects BAB integrity, and the effect subsides within 7 days. Eyelid surgery does not affect BAB.Eye advance online publication, 13 February 2015; doi:10.1038/eye.2014.337.
... Another aspect of localized tissue activation in TAO relates to the underlying mechanisms through which professional immune cells such as T and B cells, monocytes, and mast cells might be trafficked to the orbit. An array of chemoattractants expressed by orbital fibroblasts, endothelial cells, and vascular smooth muscle could help explain the infiltration of CD4 + and CD8 + T cells that have been identified in affected tissues [35][36][37][38] . These include molecules that are classified as chemokines by virtue of their harboring signature protein sequences and others that lack these motifs but function similarly. ...
Thyroid-associated ophthalmopathy (TAO) is a vexing and undertreated ocular component of Graves disease in which orbital tissues undergo extensive remodelling. My colleagues and I have introduced the concept that fibrocytes expressing the haematopoietic cell antigen CD34 (CD34(+) fibrocytes), which are precursor cells of bone-marrow-derived monocyte lineage, express the TSH receptor (TSHR). These cells also produce several other proteins whose expression was traditionally thought to be restricted to the thyroid gland. TSHR-expressing fibrocytes in which the receptor is activated by its ligand generate extremely high levels of several inflammatory cytokines. Acting in concert with TSHR, the insulin-like growth factor 1 receptor (IGF-1R) expressed by orbital fibroblasts and fibrocytes seems to be necessary for TSHR-dependent cytokine production, as anti-IGF-1R blocking antibodies attenuate these proinflammatory actions of TSH. Furthermore, circulating fibrocytes are highly abundant in patients with TAO and seem to infiltrate orbital connective tissues, where they might transition to CD34(+) fibroblasts. My research group has postulated that the infiltration of fibrocytes into the orbit, their unique biosynthetic repertoire and their proinflammatory and profibrotic phenotype account for the characteristic properties exhibited by orbital connective tissues that underlie susceptibility to TAO. These insights, which have emerged in the past few years, might be of use in therapeutically targeting pathogenic orbit-infiltrating fibrocytes selectively by utilizing novel biologic agents that interfere with TSHR and IGF-1R signalling.
... Recruitment and infiltration of assorted classes of immune cells in the orbit is dependent upon an abundant pool of the produced cytokines. Cytokines have important putative roles in GO development, progression, and regression and are produced by activated T-cells (mostly CD4+ cells), macrophages, B-cells, and orbital fibroblasts [48-50]. Th1 lymphocytes and their associated family of cytokines [TNF-α, interleukin 1β (IL-1β), INF- γ, IL-2, and interleukin 12 (IL-12)] [51] predominate the orbit early in the course of the disease and promote the active phase of GO. ...
Graves' disease (GD) is the most common cause of thyrotoxicosis and often involves the orbits. Graves' ophthalmopathy (GO), also known as Thyroid Eye Disease (TED), can be clinically significant and advance to sight-threatening stages. Our knowledge of the immunogenetic pathophysiology of GO is rapidly expanding. The present review is an attempt to summarize the current state of knowledge on the immunogenetics of GO. First we briefly review the epidemiology and clinical importance of GO, and then we describe in detail the macromolecular pathogenesis and finally immunogenetics of GO. Discrepancies between the results from various reports and the limitations of the available data are discussed. In particular, there is a scarcity of data from non-Asian populations. While several studies have demonstrated significant associations between polymorphisms in certain genes (especially CTLA-4, HLA-DRB-1, and TNF-α), there is a need for studies that investigate the relationship between polymorphisms and both serum and local concentrations of the resulting proteins. A complete understanding of GO susceptibility and pathogenesis has not been yet possible due to a number of important knowledge gaps that need to be filled by future research.
... 3 Higher concentra tions of IL15 were detected in sera from patients with Hashimoto thyroiditis com pared with euthyroid individuals. 4 More over, Pappa et al. 5 detected IL15 in 33% of thyroidassociated ophthalmopathy biopsies from extraocular muscle. ...
We read with great interest the News and Views article by Terry J. Smith and Raymond S.
... Histochemical examination of affected tissues demonstrates a diffuse infiltration of T and B lymphocytes, occasional macrophages, and the presence of proinflammatory cytokines (9,10). These cytokines, secreted by the infiltrating mononuclear cells as well as by the resident orbital macrophages and adipocytes, contribute to the inflammation characteristic of active GO (9,(11)(12)(13)(14)(15)(16). Other studies have shown that TSHR is expressed in orbital adipose= connective tissues (1)(2)(3)17,18) and that its expression is elevated in the GO orbit (5,6,19) and is highest in the orbits of GO patients having active disease (20). ...
Patients with Graves' ophthalmopathy (GO) have circulating autoantibodies directed against the thyrotropin receptor (TSHR) and elevated levels of the proinflammatory cytokine interleukin-6 (IL-6) in both serum and orbital tissues. We hypothesized that these autoantibodies might increase IL-6 expression and secretion in preadipocyte fibroblasts and adipocytes from patients with GO, and thus directly impact the clinical activity of the disease.
IL-6 mRNA levels were measured in cultures of GO orbital preadipocytes (n = 3) treated during adipocyte differentiation with a monoclonal stimulatory TSHR antibody (M22; 10 ng/mL), IL-6 (1 ng/mL), or TSH (10 U/L). Additionally, levels of IL-6 protein secretion were assessed after adipocyte differentiation in orbital cultures exposed to TSH or M22 for 24 or 48 hours (n = 8). IL-6 mRNA levels were also measured in orbital adipose tissue specimens from well-characterized GO patients (n = 9) and normal individuals (n = 9).
Treatment of GO orbital preadipocyte cultures with IL-6, TSH, or M22 during adipocyte differentiation resulted in increased IL-6 mRNA levels (3.1-fold, 2.9-fold, and 2.7-fold, respectively; p < 0.05). Treatment of orbital cultures with M22 or TSH after adipocyte differentiation enhanced the release of IL-6 protein into the medium at both 24 and 48 hours for TSH (mean 1.9- and 2.3-fold; p = 0.002 and 0.015, respectively) and at 48 hours for M22 (mean 2.0-fold; p = 0.005). In addition, we found mean IL-6 mRNA levels to be significantly increased in GO orbital adipose tissue specimens (10-fold; p < 0.01), primarily attributable to high levels in three of the four patients with clinical activity scores >or=5.
Both TSH and M22 increase IL-6 expression in orbital preadipocyte fibroblasts and IL-6 secretion by mature adipocytes. These results suggest that circulating TSHR autoantibodies in GO might play a direct role in the clinical activity of the disease.
Objective:
To evaluate the performance of T1 mapping in the characterization of extraocular muscles (EOMs) of Graves' ophthalmopathy (GO) patients and investigate its feasibility in assessing the response to glucocorticoid therapy in active GO patients.
Methods:
A total of 133 participants (78 active GO, 23 inactive GO, 18 Graves' disease (GD) patients, and 14 healthy volunteers) were consecutively enrolled from July 2018 to December 2020. Native T1 (nT1) and postcontrast T1 (cT1) values of EOMs were measured and compared. The variations in T1 mapping metrics of EOMs were compared pre/post glucocorticoid treatment in 23 follow-up active GO patients. Logistic regression analysis and receiver operating characteristic (ROC) curve analysis were performed.
Results:
The nT1 of EOMs in GO patients was higher than that in GD patients and healthy volunteers. The nT1 of superior rectus (SR) in active GO was higher than that in inactive GO patients, and it could be used as a potential marker of GO activity (OR: 1.003; 95% CI: 1.001, 1.004), with a diagnostic sensitivity of 86.3% and specificity of 43.7%. Meanwhile, the cT1 of SR, inferior rectus (IR), and medial rectus (MR) in inactive GO patients were higher than those in active GO patients. The nT1 of EOMs achieved sufficient diagnostic performance in evaluating the response to glucocorticoid therapy for follow-up active GO patients (AUC, 0.797; sensitivity, 71.9%; specificity, 85.7%).
Conclusions:
T1 mapping could quantitatively assess the activity of GO and the response to glucocorticoid therapy in active GO patients and may even potentially reflect the fibrosis of EOMs.
Clinical relevance statement:
T1 values can reflect the pathological status of the extraocular muscle. T1 mapping could help to quantitatively assess the clinical activity of GO and the response to glucocorticoid therapy in active GO patients.
Key points:
• Graves' ophthalmopathy patients had greater nT1 of extraocular muscles than Graves' disease patients and healthy volunteers, and nT1 of the superior rectus could be a potential marker of Graves' ophthalmopathy activity. • The cT1 of extraocular muscles in inactive Graves' ophthalmopathy patients was higher than that in active Graves' ophthalmopathy patients, and it might be associated with muscle fibrosis. • nT1 of extraocular muscles could offer sufficient diagnostic performance in evaluating the response to glucocorticoid therapy for follow-up active Graves' ophthalmopathy patients.
Background:
Graves' orbitopathy (GO) is an autoimmune driven manifestation of Graves' disease (GD) where pathogenic autoantibodies to the TSH-receptor activate orbital fibroblasts/preadipocytes in the orbital tissue to induce inflammation and extracellular matrix deposition. Since there are significant limitations to study immunological and proinflammatory mediator expression in early and during disease progression in GO patients, we used our experimental mouse model to elucidate early pathogenic processes.
Methods:
We have developed a robust mouse model of GD/GO induced by electroporation immunization of plasmid encoding human TSHR A-subunit, comprising of multiple injections over a course of 15 weeks to fully recapitulate the orbital pathology. In this study, we investigated kinetics of GO development in the model by serial analyses of immunological and cellular parameters during course of orbital inflammation.
Results:
Pathogenic anti-TSHR antibodies with thyroid stimulating properties developed early after the second immunization step with concomitant induction of hyperthyroidism. Examination of orbital tissue showed an early wave of macrophage infiltration followed subsequently by CD3+ T-cells into the orbital tissue. Examination of antigen-specific T-cell activity using recombinant human A-subunit protein showed high CD8+ T-cell proliferation during this early phase of disease onset, whereas effector CD4+ T cells and CD25+FOXP3+ regulatory T-cells were downregulated. The early phase of disease was also characterized by abundant presence of proinflammatory cytokines IFN- and TNF-. Moreover, as the disease progressed there was significant increase in browning of orbital fat tissue, which may be dependent on the pro-inflammatory milieu and/or the increased thyroid hormone levels during the established hyperthyroid status.
Conclusion:
This work revealed early infiltration of macrophages in the orbital region and induction of pathogenic anti-TSHR antibodies during disease onset in the model. This was followed subsequently by influx of CD8+ T cells specific for TSHR coupled with reduction in Tregs and substantial increase in BAT. These new insights into the development of orbital inflammation in the model have implications for testing new therapeutic regimes by targeting macrophage function during early phases of orbital inflammation in the model.
Thyroid-associated ophthalmopathy (TAO) is a disfiguring and potentially sight-threatening manifestation of autoimmune thyroid disease. Current therapy is limited to addressing the complications of the disease which include exposure keratitis, impairment of eye movements due to extraocular muscle (EOM) inflammatory involvement, and optic nerve compression with possible loss of vision. TAO is characterised by mononuclear cell infiltration, many of which are T cells, of the EOMs and/or the orbital fat/connective tissue with associated deposition of glycosaminoglycans (GAGs) in the interstitial spaces. In this thesis, the presence and distribution of the vascular adhesion molecules ICAM-1, ELAM-1, VCAM-1 and the leukocyte integrins CD11a/CD 18, CD11b/CD18, CD11c/CD18 were investigated, by immunohistochemistry, on EOM biopsies harvested from early, active and late, inactive TAO patients as well as non-TAO strabismus control subjects. Because of small biopsy size, on different EOM biopsies collected from the same groups of patients the mononuclear cell infiltrate as well as the expression and localisation of HLA-DR were also characterised immunohistochemically. Further EOM biopsies were taken from TAO patients and the infiltrating T cells were isolated and expanded in vitro with mitogen. Their phenotype was determined by FACS analysis and compared to peripheral blood-derived T cell lines, grown in vitro in the same way, from the same patient. Cytokines present in the supernatant after mitogen stimulation of the T cell lines, were assayed by ELISA techniques. Moreover, the pattern of cytokine gene expression in EOMs was studied ex vivo in biopsies taken from different TAO patients, and results were compared with the data derived from the T cell lines. With the T cell lines from two patients, proliferation assays were carried out using antigens derived from thyroid gland, EOM and a TSH-receptor preparation. In addition, the presence and localisation of GAGs on TAO and control EOM biopsies was examined by transmission electron microscopy and immunogold staining. Serum hyaluronan was measured using a radioimmunoassay in patients with TAO as well as control subjects, and urinary GAG levels assessed by photometric quantitation of hexuronic acid after reaction with carbazole. Finally, the excretion pattern of the urinary GAGs was determined by means of discontinuous electrophoresis.
Our study aimed to reveal the underlying pathologic mechanisms of thyroid-associated ophthalmopathy (TAO) by integrative transcriptomics and proteomic analysis of extraocular muscles (EOM). The study involved 11 TAO patients (clinical activity score ≤ 2) and 11 control donors. Total RNA was extracted from EOM samples of 5 TAO patients and 5 control individuals for gene microarray analysis to reveal differentially expressed genes. Concurrently, EOM samples from 3 TAO patients and 3 control individuals were lysed for quantitative proteomic analysis. Differentially expressed genes and proteins were identified, followed by functional and pathway enrichment analysis and protein-protein interaction network construction. Concordance between proteins and transcripts was examined, and functional annotations were conducted. Expressions of versican (VCAN) and lipocalin 1 (LCN1) in EOM samples from another 3 TAO patients and 3 control individuals were measured by western blotting. In total, 952 genes and 137 proteins were identified as differentially expressed, as well as 96 differentially expressed proteins without significantly changed mRNA abundance. Proteins mainly related to the composition (such as MYH1, MYH2, and MYH13) and contraction force (MYH3, MYH8, ACTN3, and TNNT1) of the muscle fibers were significantly up-regulated in EOM samples of TAO, as well as those (such as VCAN, MPZ, and PTPRC) associated with cell adhesion. In addition, differentially expressed proteins related to the components and metabolism of extracellular matrix (ECM) (such as COL1A1, COL1A2, COL2A1, VCAN, OGN, and DCN) were identified. Similarly, expressions of genes involved in cell adhesion and ECM metabolism were significantly different between EOM samples of TAO patients and controls. Western blotting verified that VCAN involved in ECM proteoglycans and diseases associated with glycosaminoglycan metabolism was markedly higher in EOM samples of TAO, whereas LCN1 was obviously decreased. In conclusion, this study demonstrated the significantly altered cellular components of EOM, muscle contraction, cell adhesion and ECM metabolism, which might be involved in the pathologic mechanisms and/or consequences of TAO.
Since the 1970s, the role of infectious diseases in the pathogenesis of Graves' disease (GD) has been an object of intensive research. The last decade has witnessed many studies on Yersinia enterocolitica, Helicobacter pylori and other bacterial organisms and their potential impact on GD. Retrospective, prospective and molecular binding studies have been performed with contrary outcomes. Until now it is not clear whether bacterial infections can trigger autoimmune thyroid disease. Common risk factors for GD (gender, smoking, stress, and pregnancy) reveal profound changes in the bacterial communities of the gut compared to that of healthy controls but a pathogenetic link between GD and dysbiosis has not yet been fully elucidated. Conventional bacterial culture, in vitro models, next generation and high-throughput DNA sequencing are applicable methods to assess the impact of bacteria in disease onset and development. Further studies on the involvement of bacteria in GD are needed and may contribute to the understanding of pathogenetic processes. This review will examine available evidence on the subject.
Graves’ ophthalmopathy (GO) is generally accepted to be an autoimmune inflammatory disorder of the extraocular muscles (EM) and the orbital fatty/connective tissue (OCT) that is closely associated with Graves’ disease (1–3).
Various names have been given to the eye signs associated with autoimmune thyroid disease—Graves’ ophthalmopathy, endocrine exophthalmos, ophthalmic Graves’ disease, and so on—but the term thyroid-associated ophthalmopathy (TAO) is now generally accepted as the best, providing recognition that the condition is not always associated with Graves’ disease. The major clinical signs are shown in Table 1 and Fig. 1. Fortunately TAO does not cause problems of diplopia or loss of visual fields in the majority of patients, but the impact of having bulging eyes and periorbital swelling on the patient’s sense of well-being is often underestimated, and even the grittiness and discomfort that are common and among the early features of TAO prove irksome to many patients with Graves’ disease.
It is now fully accepted that Graves’ ophthalmopathy is an orbital autoimmune disease closely related to Graves’ disease and, less commonly, found in autoimmune hypothyroidism (1). These clinical features have led to the hypothesis, discussed at length elsewhere in this volume, that ophthalmopathy is the result of an autoimmune response against one or more orbital autoantigens which are shared by the thyroid. Such antigenic cross-reactivity would explain the known clinical associations and temporal features of the disease but it should still be recognized that any such cross-reactivity could be at the T cell rather than B cell level, and that, so far, this is still not a firmly established fact. Indeed, it remains the case that establishing the nature of autoantigen(s) responsible for ophthalmopathy is the Holy Grail of research in this area, as knowledge of the autoantigen responsible for an autoimmune disease is key to any experiments designed to map pathogenic epitopes or to any autoantigen-based therapeutic endeavors.
Objective: Thyroid associated ophthalmopathy (TAO) is a kind of immunopathogenic disorder. The immunocompetent cells may be important mediators in the local pathogensis of TAO. The distribution of immunocytes in extraocular muscle (EOM) of eye with TAO was studied to explore the pathogenic mechanism of TAO. Methods: Eight EOM specimens were collected from 8 patients with TAO during squint surgeries. The specimens in controls were obtained from healthy corpse eyes. The cell distributions of immunocytes, including macrophages, HLA-DR positive cells, T cells and B cells were analyzed quantitatively using SP immunohistochemical method. Results: Abundant macrophages, HLA-DR positive cells and T cells were found in extraocular muscle of TAO. Whereas B cells were absent in all specimens. There was a significant increase in macrophages in TAO compared with the control(P < 0.01). In extraocular muscle of TAO, macrophages were more two times than control tissues (97 ± 10.15/mm 2 in TAO,48 ± 7.17/mm2 in control). CD4+ T cells with brown color in cytoplast were evident in TAO specimens and CD8+ T cells in control tissues. Conclusion: Macrophages is probably involved in the pathogenesis of TAO. Early intervention of immunosuppressive therapy of anti-macrophage may influence the sequel caused by extraocular muscle fibrosis in TAO.
To explore the role of CD4(+)CD25(+)Foxp3(+)Treg/CD4(+)IL-17A(+)Th17 cells and the related cytokines in Graves' ophthalmopathy.
Based on clinical activity scores (CAS), we divided patients with untreated Graves' ophthal- mopathy into active group (AGO group with CAS≥3 (15 cases) and non-active group (NGO group) with CAS<3 (15 cases), with another 15 patients with untreated Graves' disease free of eye symptoms (GD group) and 15 normal subjects as controls. Peripheral venous blood Treg/Th17 cell ratio was determined using flow cytometry. RT-PCR was used to detect the mRNA expression levels of Treg-specific transcription factor Foxp3 and Th17-specific transcription factor RORγt. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of Th17 cell-related cytokines (IL-17A, IL-23, and IL-6) and Treg-related cytokines (TGF-β, IL-10, and IL-35).
Compared with the normal subjects, the patients in GD, NGO, AGO groups all showed significantly increased Th17 cell count (P<0.05), which was the highest in AGO group. RT-PCR results revealed significantly increased RORγt in GD, NGO, and AGO groups, also the highest in AGO group. Serum IL-17A, IL-23, and IL-6 levels all showed significant increments in GD, NGO, and AGO groups (P<0.05), especially in AGO group. Among the Treg-related cytokines, TGF-β and IL-35 levels decreased (P<0.05) but IL-10 increased significantly (P<0.05) in GD, NGO, AGO groups.
Decreased immunosuppressive capacity of Treg cells can be an important factor in the pathogenesis of Graves' ophthalmopathy. Th17 cells may also participate in the occurrence and progression of Graves' ophthalmopathy and can serve along with related cytokines as novel indicators of the disease activity. Impaired Treg/Th17 balance may importantly contribute to the occurrence of Graves' ophthalmopathy.
: The pathophysiology of thyroid eye disease (TED) is complex and incompletely understood. Orbital fibroblasts (OFs) seem to be the key effector cells that are responsible for the characteristic soft tissue enlargement seen in TED. They express potentially pathogenic autoantigens, such as thyrotropin receptor and insulin-like growth factor-1 receptor. An intricate interplay between these autoantigens and the autoantibodies found in Graves disease may lead to the activation of OFs, which then leads to increased hyaluronan production, proinflammatory cytokine synthesis, and enhanced differentiation into either myofibroblasts or adipocytes. Some of the OFs in TED patients seem to be derived from infiltrating fibrocytes. These cells originate from the bone marrow and exhibit both fibroblast and myeloid phenotype. In the TED orbit, they may mediate the orbital expansion and inflammatory infiltration. Last, lymphocytes and cytokines are intimately involved in the initiation, amplification, and maintenance of the autoimmune process in TED.
: A 43-year-old man with a high-grade glioma involving the cerebellar nodulus showed a near-complete suppression of periodic alternating nystagmus (PAN) in the lateral decubitus position to either side. This positional modulation of PAN is consistent with suppression of the velocity storage mechanism by head position changes (tilt dumping) and is supportive of the role of the velocity storage mechanism in generating PAN.
About 5% of patients with Graves' disease (GD) develop Graves' orbitopathy (GO) of sufficient severity to require treatment. GD is an autoimmune condition caused by thyroid stimulating antibodies (TSAB) mimicking the action of TSH, although the mechanisms leading to loss of tolerance to the TSHR remain unknown. In GO, tissue remodeling increases the volume of the orbital contents by several mechanisms including adipogenesis and overproduction of glycosaminoglycans (GAGs). The increased volume produces proptosis, chemosis and increased intraocular pressure. GO is also an autoimmune disease and the timing of its onset (relative to GD) suggests a thyroid/orbit shared antigen. Data in favour of the antigen being the TSHR include demonstration of its increased expression during adipogenesis and the fact that the most severe GO occurs in GD patients having the highest TSAB titres. Evidence against its role are patients with euthyroid GO (devoid of TSAB) and its widespread expression. However, recent reports demonstrate a direct role for TSHR autoantibodies in GAG production, but it is likely that other antigens, such as IGF-1R, may also contribute to pathogenesis. In vitro models have demonstrated the heterogeneity of orbital fibroblasts, some destined for adipogenesis and/or secretion of GAGs in response to stimulation by inflammatory cytokines or patient immunoglobulins. GO may be exacerbated by e.g. PPARγ agonists, but PPARγ antagonists (inhibit adipogenesis) may provide novel treatment options. Of interest, B cell depletion, which does not consistently reduce TSAB levels, also shows some promise but further adds to the puzzle of the relevance of the TSHR.
Graves’ eye disease is characterized by a chronic round cell infiltrate of the retrobulbar tissues (sometimes of focal character and organized in T cell zones and B cell follicles1) and a very strong association with Graves’ thyroid disease. The latter is an accepted autoimmune disease and caused by autoantibodies (Abs) against the TSH-receptor (TSH-R), which stimulate thyrocytes to grow and to produce excessive amounts of thyroid hormones. On the basis of its histology and the association with Graves’ thyroid disease it has for long been the idea that Graves’ eye disease is of autoimmune character too, and that the TSH-R is an important antigen also in the pathogenesis of the eye signs. This chapter tries to summarize recent developments in endocrine autoimmunity in general and Graves’ ophthalmopathy in particular.
The most common cause of hyperthyroidism is Graves'' disease, which represents a typical example of an organ-specific autoimmune condition. The exact triggers for the disease remain unknown, but are likely to involve a complex interaction between multiple environmental factors in a genetically predisposed individual. The main feature of the condition is the presence of thyroid-stimulating antibodies, which activate the thyroid- stimulating hormone receptor, resulting in hyperthyroidism. These antibodies may also be involved in the extrathyroidal complications of the disease. The recent generation of thyroid-stimulating antibodies in animal models and the isolation of monoclonal thyroid-stimulating antibodies from a patient with Graves'' disease should allow the detailed study of thyroid-stimulating antibodies--thyroid-stimulating hormone receptor interactions. This will help to shed more light on disease pathogenesis and may offer new treatment strategies in difficult cases, particularly in patients with extrathyroidal complications.
The pathogenesis of Graves′ disease, an autoimmune thyroid disorder, remains incompletely understood. Increasing clinical
and experimental evidence shows that genetic predisposition, disturbances of the immune system function and environmental
factors affecting thyroid gland activity appear to play crucial roles. Graves′ disease is characterized by hyperthyroidism
and/or ophthalmopathy related to retroorbital tissue infiltration by autoreactive T-lym-phocytes (1). The hallmarks of the disorder, which have been considered to be responsible for ophthalmopathy, hyperthyroidism, acropachy,
pretibial derm-opathy, and goiter are factors such as circulating antibodies against thyroid autoantigens (mainly to thyrotropin
[TSH] receptor) (2,3), association with immune response (HLA-DR) genes (4–8), and an intermittent clinical course of exacerbations and remissions. It has been also proved that in Graves′ disease autoantibodies
are capable of stimulating the synthesis of hormones in thyrocytes via the central autoimmune target TSH receptor, which has
been cloned and sequenced (9). Most patients with Graves′ disease are hyperthoid. The availability of free thriiodothyronin (fT3 ), free thyroxin (fT4 ), and highly sensitive TSH techniques has vastly simplified the diagnosis of the early subclinical form of hyperthyroidism
(7). Development of TSH receptor autoantibody assays (10) has enhanced the ability of the clinician to identify subtle presentation of this autoimmune thyroid disease (7).
Graves' disease is the most frequent cause of hyperthyroidism, affecting mainly young aged women, with an etiology of autoimmune basis. One of its manifestations, Graves' ophthalmopathy whose pathophysiology is unknown, represents one of the greatest therapeutic challenges in these patients, because they require aggressive treatment with steroids and multiple subsequent reconstructive surgeries in certain cases. It also represents a high burden to the health system. Drugs targeting B cells have been very effective for many autoimmune diseases. Rituximab is a murine humanized monoclonal antibody against CD20 + cells currently being studied in various autoimmune diseases including Graves' disease. The objective of this paper is to expose possible mechanisms by which rituximab could act in both hyperthyroidism and ophthalmopathy of Graves' disease, as well as the experience with its use acquired so far. The employment of rituximab in recently diagnosed patients or with mild ophthalmopathy is questionable with the evidence available today however, we think that it may have a role in refractory cases or those who have a contraindication for steroid use.
Thyroid-stimulating hormone receptor (TSHR) stimulating autoantibodies are associated with Graves' ophthalmopathy (GO), the orbital manifestation of Graves' disease (GD). TSHR autoantibody levels and orbital TSHR expression levels correlate positively with GO disease activity. Platelet-derived growth factors (PDGF) are increased in GO and potently activate orbital fibroblast effector functions. We investigated the possible relationship between PDGF and TSHR expression on orbital fibroblasts and how that influences the immunopathological effects of TSHR autoantibodies on orbital fibroblast activity.
Orbital fibroblasts were stimulated with PDGF-AA, PDGF-AB, and PDGF-BB, and TSHR expression was determined by flow cytometry. Stimulatory effects of bovine TSH and GD immunoglobulins on orbital fibroblasts (with or without PDGF-BB preincubation) were determined by IL-6, IL-8, chemokine (C-C motif) ligand (CCL)-2, CCL5, CCL7, and hyaluronan ELISA. The TSHR blocking antibody K1-70 and the cAMP inhibitor H89 were used to determine involvement of TSHR signaling.
PDGF-AB and PDGF-BB stimulation increased TSHR expression on orbital fibroblasts, whereas PDGF-AA did not. Furthermore, stimulation with bovine TSH and immunoglobulins from GD patients induced IL-6, IL-8, CCL2, and hyaluronan production by orbital fibroblasts, and PDGF-BB preincubation enhanced this response of orbital fibroblasts. Blocking studies with a TSHR blocking antibody and a cAMP inhibitor inhibited these effects, indicating the involvement of TSHR signaling and thus of TSHR stimulating autoantibodies herein.
These findings indicate that PDGF-B containing PDGF isoforms amplify the immunopathological effects of TSHR-stimulating autoantibodies in GO patients by stimulating TSHR expression on orbital fibroblasts.
Platelet-derived growth factors (PDGF) are regulators of fibroblast activity that may be involved in the pathophysiology of Graves' ophthalmopathy (GO). We unraveled the expression and origin of PDGF family members in GO orbital tissue and investigated the effect of PDGF isoforms on IL-6 and hyaluronan production and proliferation by orbital fibroblasts.
PDGF-A, PDGF-B, PDGF-C, PDGF-D, PDGF-Rα, and PDGF-Rβ expression was determined by real-time quantitative PCR and PDGF-A and PDGF-B protein expression was determined by Western blot in orbital tissues. Orbital tissues were immunohistochemically stained for PDGF-A and PDGF-B expression, together with stainings for T cells, monocytes, B cells, macrophages, and mast cells. Effects of PDGF-AA, PDGF-AB, and PDGF-BB on orbital fibroblast proliferation and IL-6 and hyaluronan production were examined. Finally, effects of PDGF-BB- and PDGF-AA-neutralizing antibodies on IL-6 and hyaluronan production in GO whole orbital tissue cultures were tested.
GO orbital tissue showed increased PDGF-A and PDGF-B mRNA and protein levels. Increased numbers of PDGF-A- and PDGF-B-positive monocytes, macrophages, and mast cells were present in GO orbital tissue. PDGF-BB stimulated proliferation and hyaluronan and IL-6 production by orbital fibroblasts the most, followed by PDGF-AB and PDGF-AA. Finally, in particular imatinib mesylate and PDGF-BB-neutralizing antibodies reduced IL-6 and hyaluronan production by whole orbital tissue cultures from GO patients.
In GO, mast cells, monocytes, and macrophages may activate orbital fibroblasts via secretion of especially PDGF-AB and PDGF-BB. Preclinical studies with whole orbital tissue cultures show that blocking PDGF-B chain containing isoforms can be a promising treatment for GO.
Cytokines are an extraordinarily diverse group of molecules, with pleiotropic and often overlapping effects. They are crucial to the autoimmune response, and, in particular, regulation of CD4+ and CD8+ T-cell function depends on the balance of cytokines produced during an immune response. It is also now clear that cytokines are produced by a wide array of cells, including the thyroid follicular cells (TFCs). Intrathyroidal lymphocytes produce a heterogeneous pattern of cytokines and we have summarized the likely effects of these. In Graves' disease, TFCs can themselves express immunologically important molecules as the result of cytokine stimulation and these could contribute to the perpetuation of the autoimmune process. In addition, cytokines have a number of generally inhibitory effects on thyroid hormone production which would tend to counter the stimulatory effects of thyroid-stimulating hormone receptor antibodies in Graves' disease.
G raves' ophthalmopathy, also called Graves' orbitopathy, is a potentially sight-threatening ocular disease that has puzzled physicians and scientists for nearly two centuries. 1-3 Generally occurring in patients with hyperthyroidism or a history of hyperthyroidism due to Graves' disease, Graves' ophthalmopathy is also known as thyroid-associated ophthalmopathy or thyroid eye disease, because it sometimes occurs in patients with euthyroid or hypo-thyroid chronic autoimmune thyroiditis. The condition has an annual adjusted in-cidence rate of 16 women and 3 men per 100,000 population. 4 This review explores the perplexing relationship between Graves' ophthalmopa-thy, hyperthyroidism, and thyroid dermopathy, the associated skin condition. I ex-amine clinical features, histologic findings, and laboratory studies, with an empha-sis on mechanisms that could be targeted in the development of new treatments for this debilitating disease.
Graves' disease is the most common cause of hyperthyroidism and is frequently complicated by ophthalmopathy, which can be a debilitating component of the disease, resulting in impaired quality of life. The management of Graves' disease aims to restore the patient to a euthyroid state and minimise the extent of extrathyroidal manifestations such as ophthalmopathy. Management options include medical therapy, radioactive iodine (RAI) and surgical intervention in the form of thyroid resection. Graves' ophthalmopathy (GO) is often resistant to or even aggravated by medical therapy and RAI, both of which have unpredictable relapse rates. The extent of thyroidectomy in the management of benign thyroid disease remains controversial. Total thyroidectomy is being increasingly favoured as the procedure of choice for treatment of benign thyroid disease. Recent study has indicated that it can be performed with minimal complications at a rate comparable to the less radical subtotal thyroidectomy. The predictable outcome and lack ofdisease recurrence make it an attractive option for benign conditions such as Graves' disease. In addition, there is increasing evidence that total thyroidectomy may have a beneficial effect, inducing an improvement in eye signs and symptoms in cases of GO. This review discusses the indications for surgical intervention in Graves' disease, with specific focus on the extent of resection and the relative merits of subtotal and total thyroidectomy in patients with GO.
T lymphocyte chemotactic factor (TCF) was purified to homogeneity from the conditioned media of phytohemagglutinin-stimulated human blood mononuclear leukocytes by a sequence of chromatography procedures. The amino-terminal amino acid sequence of the purified TCF showed identity with neutrophil-activating protein (NAP-1). Both TCF and recombinant NAP-1 (rNAP-1) were chemotactic for neutrophils and T lymphocytes in vitro supporting the identity of TCF with NAP-1. Injection of rNAP-1 into lymphatic drainage areas of lymph nodes in Fisher rats caused accelerated emigration of only lymphocytes in high endothelial venules. Intradermal injection of rNAP-1 caused dose-dependent accumulation of neutrophils and lymphocytes.
Graves' ophthalmopathy is an autoimmune condition characterized by T cell infiltration of the retrobulbar tissue. Phenotypic and functional analysis of these infiltrating cells may provide insight into the pathogenesis of the disease. IL-2-responsive cells were therefore grown out of the retrobulbar tissue from two patients with severe Graves' ophthalmopathy undergoing orbital decompression surgery, and six T cell lines were established and characterized. They consisted predominantly of CD8 + CD45RO+ cells and secreted IL-4, IFN-gamma, and IL-10 upon activation. When screened for their antigen reactivity, all lines proliferated in response to stimulation with autologous retrobulbar fibroblasts in an HLA class I-restricted manner, but did not recognize autologous peripheral blood mononuclear cells, crude eye muscle extract, allogeneic cells, or purified protein derivate of Mycobacterium tuberculosis. In contrast, PBMC from the same patients responded readily to purified protein derivate of Mycobacterium tuberculosis and allogeneic PBMC, but did not recognize autologous fibroblasts. Interestingly, only one of the six retrobulbar T cell lines displayed cytotoxicity towards its specific target cell population. These results suggest that the retrobulbar fibroblasts are a major T cell target in Graves' ophthalmopathy. Pronounced cytokine production in the absence of target cell cytotoxicity may explain fibroblast proliferation, glycosaminoglycan secretion, and secondary eye muscle enlargement in this condition.
Background:
Scleroderma (systemic sclerosis) is a fibrotic disease characterized by an uncontrolled tissular accumulation of collagen. Several cytokines have been implicated in the fibroblast activation leading to fibrosis. For instance, we have previously demonstrated that interleukin-4 (IL-4) is a potent activator of collagen synthesis in fibroblast cultures. In this study, using immunocytochemical methods and in situ hybridization, we investigated the expression of IL-4 in normal and scleroderma skin and fibroblast cultures.
Observations:
Immunocytochemical studies with anti—IL-4 antibody were performed on biopsy specimens from 9 patients with normal skin and 11 patients with scleroderma. The label was intense or strong in 8 of the 11 scleroderma skin specimens, whereas it was negative or faint in 8 of the 9 normal skin specimens (P<.01). In situ hybridization demonstrated a significant increase of the number of IL-4 messenger RNA grains in scleroderma skin compared with normal skin (3.1± 1.5 [mean±SD] vs 0.8±0.7; P<.001). A strongly positive labeling with the anti—IL-4 antibody was found in the 4 scleroderma fibroblast cultures, whereas it was negative in the 5 fibroblast control cultures (P<.05).
Conclusions:
Our results demonstrate that IL-4 is strongly expressed in the dermis of a large majority of patients with scleroderma and might be synthesized by scleroderma fibroblasts. We suggest that IL-4 is one of the cytokines implicated in the early steps of the fibrotic process.Arch Dermatol. 1996;132:802-806
Histologic examination of the retroocular connective tissues in Graves' ophthalmopathy (GO) reveals lymphocytic infiltration and an accumulation of glycosaminoglycans (GAG), hydrophilic macromolecules produced locally by fibroblasts. We studied the in vitro effect on fibroblast GAG production of several cytokines and growth factors likely to be secreted by these activated lymphocytes or macrophages. Cultures were established from retroocular connective tissue, extraocular muscle perimysium, and pretibial skin obtained from patients undergoing orbital decompression or eye muscle surgery for severe GO and from normal individuals. Confluent cultures were treated with one of the compounds and labeled with [3H]glucosamine or [35S]sulfuric acid for quantitation of [3H]GAG or [35S]GAG accumulation. Of the various compounds examined, only interleukin-1 (IL-1) and transforming growth factor (TGF)-beta significantly stimulated [3H]GAG accumulation in a dose- and time-dependent fashion. There was no difference in sensitivity to the GAG-stimulating effect of IL-1 or TGF-beta between fibroblasts from the four anatomical sites studied or between normal and GO patient fibroblasts. In conclusion, both IL-1 and TGF-beta are potent stimulators of [3H]GAG accumulation by retroocular connective tissue and perimysial fibroblasts, as well as by fibroblasts from the dermal sites studied. Stimulation of GAG production by these cytokines, released from lymphocytes or macrophages infiltrating the retroocular space, may play a role in the accumulation of GAG in the retroocular and perimysial connective tissues in GO.
The development of a characteristic ophthalmopathy is a feature of autoimmune diseases of the thyroid. The link between the conditions has not yet been discovered, but here Jack Wall and colleagues develop the theory that an autoimmune response to a 64 kDa antigen expressed on both thyroid and eye muscle membranes is responsible for this thyroid-associated ophthalmopathy.
Extraocular muscle biopsies from normal individuals and five patients with Graves' ophthalmopathy were analyzed by immunohistochemical staining. Fibroblasts in normal extraocular muscle as well as Graves' extraocular muscles expressed HLA-class II antigens, but the muscle cells did not. There was an increase of interstitial tissue in Graves' extraocular muscles but no visible damage to the muscle cells. In four of the biopsies from Graves' patients the cellular reaction was very weak. In the fifth patient the reaction was more pronounced with macrophages predominating, but with few lymphocytes and almost equal amounts of B and T cells. We could not confirm the earlier described specific extraocular muscle antibodies in sera from Graves' patients and conclude that there seems to be an activation of orbital fibroblasts in Graves' ophthalmopathy. This may contribute to the pathogenesis of the disease.
CONNECTIVE tissue imparts cohesion, strength, and form to the organs that it invests. It is comprised of several discrete cellular and noncellular components. The fibroblast and its more specialized derivatives (such as chondrocytes and osteocytes) elaborate and release several important molecules such as fibrous proteins (collagen), attachment proteins (fibronectin), glycoproteins, and ground substance. In this article we review the now abundant evidence that fibroblasts and their products are important mediators of some peripheral expressions of thyroid disease, including myxedema, pretibial dermopathy, thyroid acropachy, and ophthalmopathy of Graves' disease. Our review concentrates predominantly on the glycosaminoglycans that comprise ground substance. The fibrous proteins are given less attention.
The early prejudice that the ground substance and connective tissue in general were rather unimportant becomes apparent in a quote attributed to Von Haller (1), who in 1757 wrote that “it is evident that a considerable part of the human body consists of matter which for so many centuries has been regarded as rubbish.” Yet it is widely appreciated in contemporary biology that ground substance and the connective tissues play a crucial role in defining the microenvironment for specialized cells to function. They are important in cell attachment and cell-to-cell communication, and they influence the manner in which cells negotiate the extracellular milieu. A brief review of the physicochemical properties of the glycosaminoglycans that compose ground substance precedes the more detailed analysis of the impact that thyroid dysfunction can have on its synthesis and disposal.
A rapid and highly sensitive technique (MAPPing: message amplification phenotyping) has been developed to simultaneously analyze the array of messenger RNAs made by small numbers of cells. The technique incorporates a micro-procedure for isolating RNA, reverse transcription of total cellular RNA to produce cDNA, and enzymatic amplification of cytokine-specific DNA fragments using the polymerase chain reaction. In this study, the technique has been applied to the analysis of cytokines produced by lymphoid cells ranging in number from a single cell to 10(6) cells. The technique should be applicable to virtually any tissue or cell type.
Contradictory results have been obtained with regards to the effect of various treatment modes on the exacerbation of Graves' ophthalmopathy, probably because the number of patients in each study was small and some studies were analyzed only in relation to one type of treatment. To circument these problems, we studied the course of Graves' ophthalmopathy after various modes of therapy for thyrotoxicosis among 537 patients with Graves' disease.
A total of 537 patients with Graves' disease were prospectively studied over an 11-year period. Thirty-one patients were lost to follow-up during the first six months after treatment and were excluded from the study. Of those remaining, 426 received one form of treatment, 79 received two kinds of therapy, and one received three kinds of therapy. Thus, surgical treatments numbered 164, radioactive iodine-131 (131I) treatments numbered 241, and medical treatments numbered 182. Ocular signs were considered improved or exacerbated by the following criteria: decrement or increment of the exophthalmos of 2 mm or more, improvement or deterioration of visual acuity, and regression or progression of extraocular muscle involvement causing diplopia.
Among patients who did not have infiltrative ophthalmopathy before treatment, there was no difference in the occurrence of posttreatment exophthalmos in the surgically, medically, and 131I-treated patients (7.1%, 6.7%, and 4.9%, respectively). The incidence and the degree of progression of ophthalmopathy in patients who already had exophthalmos before treatment were similar in the medically, surgically, and 131I-treated groups (19.2%, 19.8%, and 22.7%, respectively). Most of the progression occurred in the posttreatment euthyroid stage. The incidence of improvement of ophthalmopathy was also similar (14.1%, 12.6%, and 12.3% in the medically, surgically, and 131I-treated patients).
In conclusion, we found no influence of type of therapy for thyrotoxicosis on the clinical course of Graves' ophthalmopathy in this retrospective study of 537 patients.
We have characterized the mononuclear cell infiltrate in the extraocular muscle of three patients with Graves' disease, using antibodies which permit staining of paraffin-embedded tissue. The majority of lymphocytes, occurring in foci or interstitially, were T cells, most of which stain for CD3 or with UCHL1. T cells few, if any, stained with SN130, directed against the CD45R determinant. This suggests that these lymphocytes comprise a recently activated population within which memory cells may reside. B cells were also found but were predominantly confined to focal aggregates, and in one patient lymphoid follicles were seen. The orbital fat and connective tissue from a further two patients contained very few infiltrating cells which were mainly UCHL1-positive. Eye muscle cells did not express Ia antigens but the interstitial cells between them were Ia-positive and the vascular endothelium in four of the five specimens also stained with Ia. These results indicate that Graves' ophthalmopathy is associated with T cell, and to a lesser extent B cell, responses against the retrobulbar tissues; the extraocular muscle interstitial cells, probably including fibroblasts, may be targets of activation resulting from this infiltration.
We have sought human eye muscle membrane binding antibodies in patients with Graves' ophthalmopathy using an enzyme-linked immunoassay. Antibodies were found in patients with thyroid autoimmunity irrespective of eye signs, and binding correlated closely (r = 0.94) with binding to skeletal muscle, showing that these antibodies are not site-specific. T cells from patients with thyroid autoimmunity proliferated in response to eye muscle, but again this was not specific for eye muscle or the presence of ophthalmopathy. No single antigen was responsible for inducing proliferation. These results fail to confirm a recent report of eye muscle membrane binding antibodies in a high proportion of patients with ophthalmopathy, and suggest instead that T and B cell autoreactivity to striated muscle antigens is a frequent feature of autoimmune thyroid disease, unlikely to be directly related to eye disease.
The production of migration inhibition factor (MIF) by sensitized lymphocytes in the presence of specific antigen is an accepted in vitro test of cell mediated immunity (CMI). The MIF test has been performed in patients with untreated Graves' disease (6 patients with hyperthyroidism and exophthalmos, 9 patients with hyperthyroidism but no exophthalmos, and 9 patients with Euthyroid Ophthalmic Graves' disease) and in normal subjects, again human retroorbital muscle antigen (RMA), thyroid antigen (TA) and other organ antigens. Human thyrotrophin (TSH) was also used as an antigen, either alone or after incubation with RMA. All patients with exophthalmos (whether hyperthyroid or euthyroid) showed positive MIF to RMA. A positive MIF to RMA was also found in 1/3 of the hyperthyroid patients without exophthalmos; the remainder however did not show MIF to this antigen. All hyperthyroid patients showed a positive MIF to TA (unrelated to the presence or absence of exophthalmos). In the euthyroid patients with exophthalmos, 2/3 also showed positive MIF to TA, but the remainder were negative: in this group, there was a very good correlation between the results of the MIF test (to TA) and evidence of thyroid dysfunction. There was no MIF against TSH in any of the groups studied, nor did TSH enhance the antigenicity of RMA. These studies indicate the presence of CMI in the exophthalmos of Graves' disease. Since MIF to RMA does not correlate well with MIF to TA, it may be suggested that antigenic dissimilarity exists between RMA and TA; this implies that different populations of lymphocytes may be involved in exophthalmos as opposed to hyperthyroidism, which thus perhaps should be regarded as separate entities.
The exenterated orbital contents obtained post mortem from a 47-year-old man who had an eight-month history of treated hyperthyroidism and a two-month history of visual complaints were examined histopathologically, immunohistochemically, and by electron microscopy. All of the extraocular muscles were massively enlarged, due to early diffuse endomysial fibrosis, mucopolysaccharide deposition, and a predominantly perivascular lymphocytic and plasmacytic infiltration. Histochemical stains revealed that the intramuscular mucopolysaccharides were weakly sulfated and polycarboxylated, establishing that they were products of fibroblastic activation rather than derived from mast cells. Subsarcolemmal deposits in the myofibers were shown by electron microscopy to be collections of glycogen rosettes with intermixed lipid deposits. These inclusions were interpreted as secondary phenomena due to abnormal muscle energetics from restrictions in contractility, and not as evidence of a primary degeneration of the extraocular muscle fibers themselves. The sarcomeric organization of the striated muscle cells was undisturbed, and therefore the pathologic changes occurred int he interstitial space of the endomysium. A monoclonal antibody specific for neurofilaments (antibody 4.3 F9) revealed, in comparison with normal controls, a preferential loss of large-type axons of the proximal segment of the orbital portion of the optic nerve as well as in the intracanalicular portion. The axonal loss was associated with a mildly increased number of astrocytes as demonstrated by an antibody against glial fibrillary acidic protein. The meninges of the optic nerve, the orbital fat, and the tendons of the extraocular muscles were uninflamed. The foregoing findings are compatible with a compressive optic neuropathy mediated by the massively swollen extraocular muscles impinging upon the optic nerve at the orbital apex.
ALTHOUGH the association of exophthalmos with goiter was recognized as early as the twelfth century (1), the modern focus on the constellation of hyperthyroidism, diffuse goiter, and endocrine ophthalmopathy is best attributed to the work of Caleb Parry (1825) (2), Robert Graves (1835) (3), and Carl von Basedow (1840) (4). Since their early descriptions a plethora of reports have been published detailing the clinical and histologic features of endocrine ophthalmopathy and coining a variety of colorful synonyms and eponyms (5, 6). There remained, however, a number of important unanswered questions. These deal with the nature of the inciting factor, the pathogenesis of the disease, the unique and often asymmetric involvement of extraocular muscles, the relationship between the eye disease and the thyroid abnormality, and the most rational and efficacious methods of treatment. We will review the clinical, anatomic, histologic, and immunologic features of endocrine ophthalmopathy particularly as they apply to...
Our concepts and understanding of the etiology, evolution, and propagation of Graves' ophthalmopathy have become much more sophisticated that they were 10 years ago. Given our current state of knowledge, the following scheme for the pathogenesis of Graves' ophthalmopathy can be proposed. Circulating T cells in patients with Graves' disease, directed against an antigen on thyroid follicular cells, recognize antigenic epitopes that are shared by tissues contained in the retroorbital space. Of the cell types residing in these tissues, fibroblasts are most likely to act as both target and effector cells of the retroorbital immune process. This includes those fibroblasts present in the perimysium of extraocular muscles, which do not appear to be immunologically different from fibroblasts located in the retroorbital connective tissue. By contrast, convincing evidence implicating the human extraocular myocyte itself (rather than the tissue conglomerate of extraocular muscle) as a primary target in GO remains to be demonstrated. Together with adipocytes, fibroblasts may also serve as target and effector cells in pretibial myxedema. How autoreactive T cells escape deletion by the immune system and come to be directed against a self-antigen presented by cells residing in the thyroid gland and extrathyroidal locations is unknown. T cells are recruited to and infiltrate the orbit via certain adhesion receptors, which may also play a costimulatory role in T cell activation and facilitate antigen recognition. Analysis of variable region gene usage of the T cell antigen receptors in retroorbital T cells of patients with active GO reveals limited variability, suggesting that antigen-driven selection and/or expansion of specific T cells may occur early in the evolution of GO.(ABSTRACT TRUNCATED AT 250 WORDS)
Vassart, 1990). In particular, in Graves’ disease it seems clear that the pathogenesis is adequately explained by the presence of autoantibodies to the TSH receptor (TSHR) which mimic TSH by inhibiting hormone binding (TBII) and/or stimulating CAMP accumulation (TSAB) leading to hyperthyroidism and goitre. The fact that neonates, born to mothers with Graves’ disease, experience transient hyperthyroidism
The pathogenesis of thyroid-associated ophthalmopathy is autoimmune. The questions to which answers are eagerly awaited are the identification of the autoantigen(s) and the definition of the autoimmune processes (cellular or humoral) responsible. Cellular and humoral immune responses and modulation by cytokines, against orbital tissues have been described. A link between the thyroid and the orbit seems inevitable, possibly in the form of a cross-reactive antigen, and top of the list of candidate antigens is the TSH receptor. Optimal treatment of TAO necessitates careful assessment. Thoughtful planning and timing and choice of intervention with conventional therapies, can lead to satisfactory results in the majority of cases. In addition to treating the severe complications, such as optic neuropathy, corneal exposure and muscle misalignment, corrective surgery to reconstruct the appearance of the patient's eyes should be made available.
Leukoregulin, a 50-kDa glycoprotein lymphokine, can regulate the extracellular matrix in dermal fibroblasts. Here we investigate the effects of leukoregulin on the synthesis of glycosaminoglycans in human orbital fibroblasts. We demonstrate that leukoregulin enhances the incorporation of [3H]glucosamine into glycosaminoglycans. The effect is dose dependent in the concentration range tested (0.1-2 U/ml), is maximal at 1 U/ml, and is time dependent. [3H]glycosaminoglycan accumulation is enhanced 7.67 +/- 1.23-fold (SE, n = 7) in orbital fibroblast strains. Pulse-chase studies indicate that this enhanced accumulation is not a result of a decreased rate of macromolecular degradation. Radiolabeled material induced by leukoregulin is sensitive to Streptomyces hyaluronidase digestion. Dexamethasone (10(-8) M) and cycloheximide (10 micrograms/ml) can block the cytokine's stimulation of hyaluronan synthesis. [35S]sulfate incorporation into glycosaminoglycan is unaffected by leukoregulin. In dermal fibroblasts, leukoregulin increased hyaluronan synthesis 3.66 +/- 0.37-fold (n = 5 strains, P < 0.02 compared with orbit). The increase in hyaluronan synthesis in orbital fibroblasts is substantially greater than that observed previously with other cytokines, making leukoregulin a candidate molecular trigger in Graves' ophthalmopathy.
In Graves' ophthalmopathy (GO), retrobulbar connective tissue is infiltrated by T cells whose role in the pathogenesis of the disease was investigated in the present work. The aims included firstly to characterize subsets of blood lymphocytes and of sessile lymphocytes cloned from a retroorbital tissue specimen. Second, in counterstimulation assays, the ability of patients' T cells to influence cultivated retrobulbar fibroblasts and in turn the enhancement of lymphocyte proliferation by retrobulbar fibroblasts was investigated. Blood lymphocytes of 16 GO patients and 12 controls isolated by density gradient centrifugation and retrobulbar fibroblasts obtained from orbital decompression were alternately exposed to irradiation of 4000 rad (to suppress proliferation of either cell type), then cocultivated for 48 h. Subsequently, the cells (250,000 lymphocytes, 5000 retrobulbar fibroblasts) were incubated with [3H]thymidine for 24 h. A stimulation index representing the degree of proliferation in comparison with a control was determined. Screening of 62 retrobulbar lymphocyte clones by cell-ELISA revealed a CD4/CD8 ratio of 8.2, contrasting with a normal ratio of 2.1 in peripheral lymphocytes (as obtained by FACS analysis). Incubation of patient's lymphocytes with autologous retrobulbar fibroblasts resulted in a markedly elevated stimulation index (9.7) compared to incubation of lymphocytes with retrobulbar fibroblasts from controls (2.5). In another assay, the degree of stimulation amounted to 2.2 vs. -0.8. Retrobulbar fibroblasts were stimulated twice as much by lymphocytes of the same patient compared to retrobulbar fibroblasts from controls. The autologous reactions demonstrated suggest the presence of autoreactive T lymphocytes in GO patients directed against retrobulbar fibroblasts, which, in addition, are stimulated by these lymphocytes.
A role for IgA autoantibodies in Graves' ophthalmopathy is suggested by the presence of immunoglobulins of this class in Graves' orbital tissue, as detected by immunohistochemistry. We, therefore, investigated the possibility of using the polymerase chain reaction (PCR) to amplify IgA immunoglobulin genes from plasma cells infiltrating Graves' eye tissue. Template cDNA was reverse-transcribed from orbital muscle (M) mRNA of one patient (#7) and from orbital connective tissue/fat (F) mRNA of two patients (#1 and #7), both undergoing surgery for exophthalmos because of severe infiltrative ophthalmopathy. Preliminary studies to establish the PCR procedure were performed for kappa light chain DNA amplification. With the very small amount of orbital tissue template available, the sensitive "hot start" modification of the PCR was necessary to amplify significant amounts of kappa light chain DNA. Using this procedure, IgA heavy chain DNA was amplified from both connective tissue/fat (F7) and muscle (M7) cDNA of patient #7. The DNA yield was less for IgA than for IgG using the same template. There was no significant IgA (or IgG) DNA product using the connective tissue/fat cDNA of patient #1. While not implying that IgA-infiltrating plasma cells are specific for Graves' orbital tissue, our studies nevertheless demonstrate the feasibility of amplifying the genes coding for IgA antibodies from Graves' orbital tissue plasma cells. Expression of these immunoglobulin genes in future studies will make it possible to determine the antigen specificity of the antibodies expressed by Graves' orbital tissue plasma cells.
The availability of high affinity antibodies to the human TSH receptor (TSHR) would help in defining its functional domains, but this requires the production of pure receptor as immunogen. We have expressed the extracellular domain (ECD) of the TSHR (residues 21-414) as a fusion protein with maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The major protein in an electrophoretically separated, crude bacterial lysate had a molecular mass of 89 kDa, in agreement with the size predicted for the MBP-ECD fusion product. Its identity was confirmed by Western blotting in which it was recognized by two polyclonal antibodies to synthetic peptides of the TSHR and an anti-MBP. Following purification on an amylose column, 15 mg pure MBP-ECD per litre of culture were produced, which was 5% of the total bacterial protein. Following extensive dialysis in a buffer which produces slight denaturation, MBP-ECD was cleaved with factor Xa. The identity of each protein was confirmed by Western blotting. To investigate the possibility of using the fusion protein as an immunogen we produced rabbit polyclonal antibodies to the ECD which were able to produce immunofluorescent staining of Chinese hamster ovary cells that expressed the TSHR, and revealed a protein of 95 kDa in Western blots of the same cells, in addition to a protein of 55 kDa. Only the protein of 55 kDa was detected in Western blots of human thyroid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Cytokines have a central role in the generation of an autoimmune response and can directly affect the target organ. In Graves' disease, both the infiltrating mononuclear cells and the thyroid follicular cells produce certain cytokines, but the relative contribution of each is unclear, and there are conflicting data on the exact profile of cytokines expressed within the thyroid. To clarify these issues, we used the method of reverse transcription-polymerase chain reaction amplification to analyze cytokine gene expression by intrathyroidal lymphocytes (ITL) and purified thyroid follicular cells (TFC) from six patients with Graves' disease. All ITL samples were positive for interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF alpha) messenger ribonucleic acids (mRNAs). Four samples were positive for IL-2 mRNA, and of these, three were also positive for interferon-gamma (IFN gamma). All TFC samples contained IL-6 and IL-8 mRNAs, even after depletion of CD3-positive T-cells. One TFC sample was additionally positive for IL-10 and TNF alpha mRNAs, and in the case of IL-10, this signal was not eliminated by CD3-positive T-cell depletion. IL-4 was not detected in any sample of ITL, TFC, or whole tissue. Semiquantitative analysis showed that the ITL fraction represented the major source of IL-6, IL-8, and TNF alpha mRNAs. By contrast, only three of five multinodular goiter samples were positive for IL-1 alpha mRNA; of these, two were also positive for IL-6, and 1 was positive for IL-8 mRNA. One multinodular goiter sample was positive for IL-8 mRNA alone, but IL-2, IL-4, IL-10, and TNF alpha mRNAS were not detected. These results suggest that although the TFC themselves may express certain cytokines, the ITL population represents the most important source of cytokine production in Graves' thyroid glands. The presence of IL-2, IFN-gamma, and TNF alpha and the absence of IL-4 mRNA in samples of ITL indicate a pattern of cytokine production that most closely resembles that of the TH1 helper T-cell subset. Given the etiological role of thyroid-stimulating antibodies in Graves' disease, the production of which is likely to depend upon TH2 helper T-cell function, it is perhaps surprising that the TH1 subset appears to predominate. It is possible that IL-10 is important in stimulating intrathyroidal autoantibody production, and this cytokine may also play a role in inhibiting cell-mediated thyroid injury in Graves' disease.
Lymphocytic infiltration of muscular and connective tissues of the retroorbital (RO) space is a histological hallmark of Graves' ophthalmopathy (GO). We have characterized some phenotypical and functional features of T cells derived from RO infiltrates of four GO patients who were submitted to orbital decompression. Fragments of RO tissue were cultured for 7 days in IL-2-conditioned medium in order to generate T cell lines of in vivo activated T cells. Phenotypical analysis of freshly isolated peripheral blood (PB) lymphocytes both from patients and four healthy controls showed a predominance of CD4+ T cells (CD4/CD8 ratios 1.9:2.5), whereas RO-derived T cell lines displayed almost equal proportions of CD4+ and CD8+ cells (CD4/CD8 ratios 0.9:1.2). RO T cell lines and PB T cells from patients and controls were then cloned using a high-efficiency cloning procedure. The phenotypical and functional features of 153 T cell clones (TCC) derived from RO infiltrates were examined and compared with those of 166 and 236 TCC derived from the PB of patients and controls, respectively. CD4/CD8 ratios ranged from 0.8-1.4 in the series of RO-derived TCC and from 1.9-2.2 in the corresponding series of PB-derived TCC. Assessment of lectin-dependent cytolytic activity showed similar proportions of cytotoxic clones in TCC derived from the PB of patients (37%) and controls (38%); most of the cytolytic TCC was CD8+. In contrast, the proportion of cytolytic RO TCC was markedly higher (106/153 = 69%), including 100% of CD8+ and the majority (59/79 = 75%) of CD4+ clones. When compared to TCC derived from the PB of both patients and controls, RO TCC showed remarkably high proportions of both CD8+ and CD4+ clones with a Th1-like cytokine profile, as documented by their ability to secrete IL-2, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), but not IL-4 or IL-5. This study provides evidence that cytolytic T cells with Th1 profile of cytokine production predominate in RO infiltrates of GO, a pattern quite similar to those previously described in thyroid infiltrates of Hashimoto's thyroiditis or Graves' disease. The peculiar cytokine secretion profile of RO T cells may be of importance in the pathogenesis of both the tissue alterations and fibrogenic process observed in GO.
Whether Graves' ophthalmopathy is the results of a cell-mediated or humoral autoimmune response is controversial. T-Lymphocytes that regulate these two mechanisms, Th1 and Th2, respectively, are characterized by the profile of cytokines that they secrete. We, therefore, investigated the spectrum of T-lymphocyte cytokines expressed in Graves' orbital tissue by means of the polymerase chain reaction using reverse transcribed mRNA as template. From three Graves' thyroid tissues (positive controls), we obtained DNA products of the predicted size for the Th1 cytokines interferon-gamma (IFN gamma) and interleukin-2 (IL-2) as well as for Th2 cytokines IL-4 and IL-5. A signal for IL-10 (Th1 and Th2) was also detected. No cytokine signals were observed in peripheral blood (negative control). Despite the presence of T-cells (CD3 delta marker) in the orbital connective tissue/fat of all five patients studied as well as in orbital muscle from one of these patients, IFN gamma reverse transcribed mRNA was notably absent from these tissues. In contrast, IL-2, IL-5, and IL-10 cDNA was present in orbital tissues from one or more patients. Of particular importance, an IL-4 signal was detected in orbital connective tissue/fat of patients 6 and 7 as well as in muscle of patient 7. The balance between IL-4 and IFN gamma determines whether an immune response is predominantly humoral or cell mediated. Our finding of IL-4, but not IFN gamma, mRNA expression in orbital tissue supports a role, at least in some patients, for humoral autoimmunity in Graves' ophthalmopathy.
Thyroid-associated ophthalmopathy (TAO) is most likely to be a T cell-mediated disease, in which cytokines released in the extraocular muscles activate fibroblasts, increasing glycosaminoglycan production. The nature of the orbital antigen recognized by the infiltrating T cells is unclear, although it is possible that there is cross-reactivity between this and a thyroid autoantigen to explain the close association with thyroid autoimmunity. We have tested the ability of human and porcine eye muscle antigen preparations to stimulate proliferation of circulating T cells from healthy subjects and patients with TAO or Graves' disease without clinical TAO. Occasional responses were seen, particularly after depletion of CD8+ T cells, and two out of 10 TAO patients responded to eye muscle proteins of 25-50 kD after fractionation of antigens on gels and subsequent elution. There was no disease-specific response of T cells to R1, R14, D1 and 1D3, recombinant proteins identified from screening an eye muscle cDNA library with sera from patients with autoimmune thyroid disease. We have also found that interferon-gamma (IFN-gamma) production by T cells from TAO patients was not stimulated by eye muscle membrane antigens or by 1D3. These results suggest that the frequency of circulating T cells responding to eye muscle antigens in TAO is low, and that several candidate orbital antigens, including the 64-kD protein 1D3, are unlikely to be important T cell autoantigens in this condition.
Graves’ ophthalmopathy (GO) is a medically incurable, chronic autoimmune process that affects the orbital tissues and is tightly linked with autoimmune thyroid diseases, most notably Graves’ disease (GD). The close clinical association between immunogenic hyperthyroidism, ophthalmopathy, and pretibial dermopathy suggests that the antigen responsible for these diverse conditions is common to thyroid gland, orbital tissues and pretibial skin.1,2 Gross examination of orbital tissues in patients with severe, active GO reveals edematous, enlarged extraocular muscle bodies in conjunction with an increased volume of orbital connective and fatty tissue (Chapter 1). Microscopically, two characteristic abnormalities are apparent: the presence of excessive amounts of hydrophilic glycosaminoglycans (GAGs), predominantly hyaluronic acid and chondroitin sulfate, and marked infiltration of the orbital connective tissue and extraocular muscles by immunocompetent cells (macrophages and T lymphocytes, few B cells). The orbital inflammatory process is likely to be driven by T-cells which, in response to an uncertain antigen, access and infiltrate the orbital space via certain adhesion molecules.1,3 Once recruited, these T cells release numerous cytokines capable of stimulating cell proliferation, GAG synthesis, and generation of new fat cells from orbital adipose precursor cells responding to adipogenic stimuli. Moreover, many of these autocrine and paracrine factors act to alter the expression of an array of immunomodulatory molecules in orbital preadipocyte fibroblasts.
Paracrine interactions between fibroblasts residing in the retro-ocular space and infiltrating lymphocytes/macrophages are thought to be of central importance in the pathogenesis of Graves' ophthalmopathy (GO). Although various roles have been suggested for interferon-gamma (IFN gamma), tumour necrosis factor-alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) in GO, their actual presence in Graves' retro-ocular connective tissue has not been demonstrated. We examined surgical specimens obtained during orbital decompression from patients with severe GO (n = 6), and from normal individuals (n = 5), for the presence of IFN gamma, TNF alpha and IL-1 alpha. We used immunohistochemical methods on frozen tissue sections and primary fibroblast cultures, and sodium dodecylsulfate polyacrylamide-gel electrophoresis of tissue extracts and tissue culture supernatants. In addition, immunohistochemical staining of tissues for characterization of the mononuclear cell infiltrates was performed. Aggregates of mononuclear cells in retro-ocular connective and fatty tissue were found in five of six GO tissue specimens, but in none of the control specimens. We detected immunoreactivity for the three cytokines (IFN gamma, TNF alpha and IL-1 alpha) in the five GO tissue specimens that contained mononuclear cell aggregates. In addition, IL-1 alpha immunoreactivity was demonstrable in primary and subsequent GO fibroblast cultures and in their supernatants. In contrast, no immunoreactivity for any of these cytokines was detected in tissue specimens, primary cultures or culture supernatants derived from normal individuals. The presence of mononuclear cell infiltrates and associated immunoreactivity for IFN gamma, TNF alpha, IL-1 alpha in retro-ocular connective tissue derived from patients with GO suggests that the previously demonstrated in vitro functions of these cytokines may indeed be operative in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
Lymphocytic and other mononuclear cell infiltration of the retro-bulbar space is observed in Graves' ophthalmopathy (GO). We investigated the antigenic character of orbital adipose/connective tissue and muscle from 21 euthyroid patients with severe GO after orbital surgery. Orbital tissue proteins were separated and recovered in soluble form by means of an electroelution technique. Twenty-two protein fractions, identified according to their molecular mass ranges, were used as antigens for orbital tissue-derived and peripheral blood T lymphocytes. Seventeen T cell lines from 6 patients were established from in vivo activated orbital T cells using interleukin-2 and anti-CD3 antibodies. T cell proliferation was measured as [3H] thymidine uptake. When screened for their reactivity to autologous adipose/connective tissue proteins, all T cell lines responded significantly to protein fractions 6-10 kDa [stimulation index (SI) = 32.9 +/- 9.8 (mean +/- SE)] and 19-26 kDa (17 +/- 5), but not to tuberculin, which was used as a control. Phenotypic analysis analysis of 10 orbital T cell lines indicated that 6 lines consisted predominantly of CD4+ cells. Incubation of a representative T cell line with allogeneic orbital protein fraction induced a very low response to protein fraction 19-26 kDa, but not to other fractions. Thyroid protein fraction 6-10 kDa also induced the proliferation of orbital T cell lines. Incubation of peripheral blood mononuclear cells with autologous orbital protein fractions gave similar results; positive responses to 6-10 and 19-26 kDa fractions were observed with orbital tissue from 12 of 14 patients (mean SI = 22 +/- 5.9 and 6.3 +/- 1.7, respectively), and positive responses were observed with orbital tissue from 3 of 4 patients to eye muscle fractions 6-10 and 19-26 kDa (13.8 +/- 6.9 and 6 +/- 2, respectively). When proteins from cultured orbital fibroblasts were used as antigens, autologous peripheral blood mononuclear cells from the 7 of the 9 patients tested responded to these 2 fractions (15.2 +/- 6.9 and 6.8 +/- 2.4, respectively), whereas a response to cultured orbital myoblasts was observed with the 19-26 kDa fraction only (SI = 8). Positive responses to abdominal adipose or muscle proteins, as controls, were not found. The demonstration of sensitized, orbital tissue-specific, T lymphocytes in the peripheral blood and orbit from patients with GO provides evidence for a role of cellular immunity in the pathogenesis of this eye disorder.
Hashimoto's thyroiditis (HT) is an organ-specific autoimmune disease in which cytokines are likely to have a role in the initiation and perpetuation of the disease. Using the reverse transcription-polymerase chain reaction (RT-PCR) we analysed the cytokine profile in four HT tissue samples. Furthermore, cell fractionation was carried out on two tissue samples and cytokine profile was studied in CD4+ and CD8+ T cells, in addition to the residual cellular infiltrate composed of CD4- and CD8- cells. Our results showed IL-1 beta, IL-4 and IL-6 mRNA expression in three out of four tissue samples, whereas IL-1 alpha, IL-2, IL-8, IL-10, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) were expressed in all tissue samples studied. Expression of IL-1 alpha and IL-1 beta was absent in both CD4+ and CD8+ subsets. However, IL-2, IL-4, IL-6, IL-8, IFN-gamma and TNF-alpha mRNA were detected in both CD4+ and CD8+ subsets. IL-10 was expressed in the CD4+ subset in one sample, whereas it was negative in both CD8+ fractions. All the cytokines studied were expressed in the residual infiltrate. These results suggest a mixed Th1 and Th2 response in HT, both of which may have an important role in the pathophysiology of the thyroid destructive process through cell-mediated cytotoxicity, and/or humoral autoimmunity manifested by autoantibody production.
IL-12, IL-13, and IL-15 are important cytokines that have not been studied previously in human autoimmune thyroid diseases. By applying RT-PCR on RNA extracted from tissue samples, we have investigated in vivo gene expression of these cytokines in multinodular goiter (MNG), Graves' disease (GD), and Hashimoto's thyroiditis (HT). In addition, in vitro studies were carried out using the transformed human thyroid cell line, HT-ori3, and primary thyroid cell cultures derived from patients with GD. These cells were used either unstimulated or stimulated for 12 h with TSH, IL-1, or interferon-gamma (IFN-gamma). IL-12 p40 gene expression was identified in 2 of 10 MNG samples, 6 of 12 GD, and 3 of 4 HT. HT-ori3 and primary thyroid cell cultures were positive for IL-12 p40 messenger RNA (mRNA) expression in both unstimulated and stimulated cell cultures. IL-13 mRNA was expressed in 2 MNG, 9 GD, and 1 HT sample. Both HT-ori3 and primary thyroid cultures expressed IL-13 after TSH, IL-1, or IFN-gamma stimulation; unstimulated primary cultures of thyroid cells, however, did not express IL-13. IL-15 gene expression was detected in 8 MNG, 8 GD, and 4 HT samples. HT-ori3 and primary thyroid cell cultures, stimulated with TSH, IL-1, or IFN-gamma, showed expression of this cytokine. Unstimulated cells showed only a weak expression. Our results indicate that the cytokine patterns in the various diseases studied are heterogeneous; that thyroid cells can express IL-12, IL-13, and IL-15 mRNA in culture, particularly after TSH, IL-1, or IFN-gamma stimulation; and that IL-15 is expressed in most of the tissue samples studied.
Quantititation of mRNA by the polymerase chain reaction
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Recombinant human TNF-a: effect on proliferation of normal and transformed cells in vitro Prostaglandin E 2 elicits a morphological change in cultured orbital fibroblasts from patients with Graves' ophthalmopathy
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