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Relative Overexpression of Macrophage-Derived Cytokines in Orbital Adipose Tissue from Patients with Graves’ Ophthalmopathy

Divisions of Pediatric Endocrinology (S.K.) and Endocrinology, Metabolism, and Nutrition (R.S.B.), Mayo Clinic, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Rebecca S. Bahn, M.D., Division of Endocrinology, Metabolism and Nutrition, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905. E-mail: bahn.rebecca{at}mayo.edu.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Graves’ ophthalmopathy (GO) is an autoimmune disorder involving the adipose and connective tissues of the orbit. The study of cytokines present in these tissues may reveal the nature of the cells and immune responses involved in GO pathogenesis. In the current study, we performed relative quantification of the expression of cytokine genes in orbital adipose tissue from patients with GO (n = 6) and normal individuals (n = 2). Real-time RT-PCR was performed using fluorescent probes and primers for cytokines including IL-1ß, IL-2, IL-4, IL-5, IL-8, IL-10, IFN-, and TNF-. Results showed IL-1ß to be the gene having the greatest fold expression increase over normal in four of six patients. TNF- was increased in all six GO patients. In addition, IL-8, IL-10, and IFN- were increased in five of six GO patients. We found no evidence of either IL-4 or IL-5 expression in any of the GO or normal samples. The increased expression of the macrophage-derived cytokines IL-1ß, TNF-, and IL-10 suggests the presence of macrophage activation and ongoing antigen presentation within the orbit in GO. In addition, the overexpression of IFN-, without evidence of IL-4 or IL-5 expression, supports the concept that cell-mediated, rather than humoral, immunity plays the predominant role in pathogenesis of this disorder.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
GRAVES’ OPHTHALMOPATHY (GO) is an autoimmune inflammatory disorder of the orbital fatty tissue/connective tissue and the extraocular muscles that is closely associated with Graves’ disease. Histological examination of the retroocular tissues from patients with GO demonstrates a diffuse infiltration of mononuclear cells, with sparse lymphoid aggregates (1, 2). The mononuclear cells present consist primarily of T lymphocytes (CD2+/CD3+), with the minority being B lymphocytes (Leu26+). Both helper/inducer (CD4+) and suppressor/cytotoxic (CD8+) subsets are present, with a slight predominance of the latter (1, 2). Other cells visualized, though less abundant, include macrophages, mast cells, and plasma cells. In addition to the inflammatory infiltration, an accumulation of glycosaminoglycans and an expansion of the orbital adipose tissue compartment are evident (1, 3, 4, 5). These changes are thought to result from the presence of particular cytokines within the orbit acting as mediators of interactions between resident fibroblasts and infiltrating or resident immunocompetent cells.

Study of the cytokines present in GO orbital tissues may suggest the nature of the cells and immune responses involved in the pathogenesis of this condition. We undertook the current study to gain additional insight into which cells, cytokine products, and immune responses might be playing the predominant roles. Real-time RT-PCR was performed on samples of orbital adipose tissue from six patients with GO and two normal individuals using fluorescent probes and primers for various cytokines potentially involved in this condition.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

Orbital adipose/connective tissue specimens were obtained from six patients who underwent orbital decompression surgery for GO (Table 1) and from two individuals with no history of Graves’ disease. These normal orbital tissues were retrieved at very early autopsy from patients whose corneas were being harvested for transplantation. All GO patients were initially diagnosed and treated for hyperthyroidism by their local physicians. Five patients had received radioactive iodine between 23 and 228 months before decompression surgery. One patient (no. 3) was taking antithyroid drugs at the time of the surgery, and another patient (no. 5) reported a remote history of antithyroid drug treatment. Five of six patients had severe inflammatory/congestive GO characterized by periorbital edema, chemosis, erythema, and eye pain with motion. The remaining patient (no. 5) had more mild, but persistent, inflammatory/congestive signs and symptoms. Time from onset of hyperthyroidism to decompression surgery ranged from 9 to 57 months (mean = 37.4 months), except in one patient (no. 5) who had onset 240 months earlier. The duration of symptomatic GO ranged from 6–35 months (mean = 20 months), except for one patient (no. 5) who first noted symptoms 180 months earlier. Three of the six patients had never received oral glucocorticoids; the remaining three had been treated with glucocorticoids in the past but had discontinued the medication several weeks before the decompression surgery. One patient (no. 2) had undergone orbital radiation therapy 23 and 29 months before the surgery. Three of the patients (no. 1–3) were current smokers. All patients were euthyroid on thyroid hormone replacement therapy at the time of orbital surgery. These studies were reviewed and approved by the Mayo Clinic Institutional Review Board and carried out according to Institutional Review Board guidelines.

Orbital adipose/connective tissue was obtained during the course of transantral orbital decompression surgery for severe GO, placed in a sterile container on saline soaked gauze, and transported at room temperature to our laboratory where it was frozen at -70 C. Normal orbital tissue was frozen immediately upon harvesting. Total RNA was isolated from orbital tissue by the RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. Complementary DNA was synthesized using 1 µg total RNA incubated with random hexamers, followed by a 100-µl reverse transcription reaction with 6.25 U Multiscribe reverse transcriptase (Applied Biosystems, Foster City, CA). Conditions used were 25 C for 10 min, 37 C for 60 min, and 95 C for 5 min.

All PCR primers and probes for IL-1, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-6R, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, IFN-, and TNF- were designed by Applied Biosystems (TaqMan cytokine gene expression plate 1, Applied Biosystems). The primers and probes were designed to avoid detection of genomic amplification. Expression of 18S rRNA was used to correct for differences in the amount of total RNA added to a reaction and compensate for different levels of inhibition during reverse transcription of RNA and during PCR. Taqman probes for the various cytokines were labeled with reporter dye FAM in the 5' end and with the quencher dye TAMRA in the 3' end. Taqman probe for 18S rRNA was labeled with the reporter dye VIC in the 5' end and with the quencher dye TAMRA in the 3' end. Amplification reactions contained 5 µl cDNA, 25 µl of the Universal Taqman 2x PCR Mastermix (Applied Biosystems), and 20 µl of Rnase-free water. Each well on the 96-well plate contained dried primers and probes for detection of one cytokine cDNA target and the endogenous rRNA control. The amplification of the endogenous control was primer limited. All reactions were performed in duplicate in the ABI PRISM 7700 Sequence Detector (Applied Biosystems) and the data pooled. The thermal cycling conditions used were: 2 min at 50 C for optimal AmpErase UNG activity, 10 min at 95 C to activate Amplitaq gold DNA polymerase, followed by 40 cycles at 95 C for 15 sec and 60 C for 1 min. Phytohemagglutinin-stimulated peripheral blood lymphocytes were used as the positive control, and the reaction mixture, without the cDNA, was used as the negative control in each run.

Calculations of relative gene expression used relative differences in the threshold cycle, the cycle of PCR at which the fluorescence reaches a given value or threshold that is in the log-linear range of amplification. Samples containing higher levels of expression of a particular gene reach the threshold value at lower cycle numbers during PCR than samples containing lower levels of expression of the same gene. Because each cycle in the PCR reaction corresponds to a 2-fold increase in PCR product, a difference in threshold cycle number of one represents a 2-fold difference in expression of a particular cytokine gene or internal control target sequence. A cytokine was considered for relative quantification and data analysis only if the threshold cycle was less than 36. If the threshold cycle was greater than 36, the cytokine was considered to be present in minimal quantities, but relative quantification in terms of fold elevation in expression was not performed. A gene was considered not to be expressed if no amplification was detected by cycle 40. Results from each GO sample were compared with the mean results from the two normal orbital specimens. Data were analyzed using Excel software (Microsoft, Redmond, WA).

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
IL-1ß, IL-6, IL-6R, IL-8, IL-10, TNF-, and IFN- genes were amplified in each of the six GO orbital tissue specimens as well as in the two normal orbital tissues (Table 2). In contrast, IL-4 and IL-5 could not be amplified in any of the GO tissues or normal orbital fat samples. IL-2 was not detectable in either normal sample but was minimally detectable in four of six GO patient samples. IL-1 ß was the cytokine having the greatest fold increase in expression in four of six patients with GO (range = 9- to 19.5-fold over the mean of the two normal tissue specimens) and was the second most elevated cytokine in the remaining two patients’ samples (3.8- and 8.4-fold; Fig. 1). The expression of TNF- was increased over normal tissue levels in all six GO patients (3.7- to 7.0-fold). IL-10, IFN-, and IL-8 were increased in five of six patients with GO (1.4- to 8.4-fold, 1.5- to 4.6-fold, and 3.0- to 13.7-fold, respectively; Figs. 1 and 2). The single patient (no. 3) showing no elevation in IFN-, IL-8, or IL-10 expression was the only patient currently taking antithyroid medication. When data were analyzed to exclude patient 3 to be able to compare mean fold elevations, we found that the cytokine having the greatest mean fold elevation overall was IL-1ß (mean 12.7-fold with SD 4.7-fold, P < 0.01; Fig. 3).

View larger version (27K): [in this window] [in a new window]   FIG. 1. Macrophage-derived cytokine gene expression in orbital tissue specimens from patients with GO. Results for IL-1ß, IL-6, IL-10, and TNF- are shown as fold elevations in individual patients’ cytokine mRNA levels relative to the mean of two normal orbital tissue specimens. A 2-fold elevation corresponds to a difference of one in RT-PCR threshold cycle number. Absence of expression data for an individual cytokine gene indicates that it was assessed, but not found to be elevated, in a particular patient’s tissue specimen.

View larger version (19K): [in this window] [in a new window]   FIG. 2. Elevations in cytokine gene expression in orbital tissue specimens from patients with GO. Results for IL-8, IL-12p, IL-15, and INF- are shown as fold elevations in individual patients’ cytokine mRNA levels relative to the mean of two normal orbital tissue specimens. A 2-fold elevation corresponds to a difference of one in RT-PCR threshold cycle number. Absence of expression data for an individual cytokine gene indicates that it was assessed, but not found to be elevated, in a particular patient’s tissue specimen.

View larger version (13K): [in this window] [in a new window]   FIG. 3. Mean fold elevations of measurable cytokine mRNA levels in orbital tissue from patients with GO (n = 5 tissue specimens for IL-1ß, TNF-, IL-8, IL-10, and IFN-; n = 4 for IL-12p35). Results are shown as mean ± SEM fold elevation in cytokine mRNA levels relative to the mean of two normal orbital tissue specimens. A 2-fold elevation corresponds to a difference of one in RT-PCR threshold cycle number.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
Cytokines are protein hormones that mediate paracrine and autocrine interactions between inflammatory cells and resident cells at sites affected by local disease processes (6). In GO, cytokines released by infiltrating lymphocytes and resident orbital fibroblasts are thought to play a key role in the initiation and propagation of the orbital autoimmune response. The potential role of macrophages and macrophage-derived cytokines in GO pathogenesis has received relatively less attention. These cells are capable of presenting antigens to the immune system via class I or class II major histocompatibility complex molecules and the delivery of essential costimulatory molecules to T cells (6). Activated macrophages are important sources of several cytokines, including IL-1, TNF-, IL-10, and IL-6, and play an important effector role in cell-mediated immune responses (7, 8, 9, 10). The concept that these cells are directly involved in GO pathogenesis is supported by histologic studies of orbital tissues in GO showing increased numbers of infiltrating macrophages, especially in early disease (5).

In the current study, we found elevated expression of the macrophage-derived cytokines, IL-1ß and TNF-, in all six GO patients. Although the majority of patients showed elevations in most of the cytokines studied, a single patient (no. 3) showed elevations in these two macrophage-derived cytokines only (Fig. 1). This particular patient was unique in that he was the only individual taking an antithyroid drug at the time of orbital decompression surgery. These compounds have been shown to have immunosuppressive properties manifest through changes in activated T-helper-like and T-suppressor-like ratios (11). It is possible that this medication modified intraorbital lymphocyte populations, although not completely inhibiting orbital macrophage activation or cytokine secretion in this patient.

The finding of increased levels of these macrophage-derived cytokines, even in tissues from patients with long-standing GO, suggests that ongoing presentation of orbital antigens by macrophages may be an important feature of the disease. However, many other cell types are found within the GO orbit, including T and B lymphocytes, orbital fibroblasts, natural killer cells, and vascular endothelial cells. Some of these cells also have the ability to secrete IL-1ß and TNF- as well as other cytokines and thus have distinct and important roles in pathogenesis (10). In addition, these cells produce other cytokines and inflammatory mediators that have pleiotropic effects on the surrounding orbital tissues. The tissue changes thus effected result in development of the clinical disease manifestations (12).

The IL-1ß genes are not constitutively expressed but can be stimulated by a variety of factors including bacterial products, complement components, and cytokines including TNF-, IFN-, and IL-1 itself. This cytokine functions as a lymphocyte activating factor (13) and induces synthesis of TNF-, IL-2, IL-6, granulocyte-macrophage colony-stimulating factor, and IL-1. This latter cytokine has been shown to stimulate in vitro proliferation of orbital fibroblasts (14, 15) and enhances the adherence of leukocytes to endothelial cells through up-regulation of intercellular adhesion molecules (16, 17, 18). We found IL-1ß to be significantly elevated in all GO patients studied, including patients with long-standing eye disease and a remote history of hyperthyroidism. In fact, the patient with the most remote onset of both hyperthyroidism and GO (240 and 180 months, respectively; patient 5) had the highest levels of this cytokine (Fig. 1). This finding suggests that macrophage activation with antigen presentation may continue in GO throughout the course of the disease.

IL-10, termed a macrophage-deactivating factor, inhibits the synthesis by monocytes of proinflammatory cytokines including IL-1, IL-6, IL-12, IFN-, TNF-, and IL-8 and induces the production of the natural inhibitors of IL-1 and TNF- (10). Thus, IL-10 exerts strong antiinflammatory activities and is thought to be one of the key factors in the homeostatic regulatory mechanisms aimed at the control of inflammation. This cytokine was found to be elevated in five of six GO patients and had the greatest fold elevations in the two patients with the longest duration of eye disease (35 and 180 months, patients 2 and 5, respectively). In addition, the patient with the highest IL-10 level (no. 5) was the patient with the least inflammatory eye disease (Fig. 1). It is possible that this cytokine functions in late-stage GO to modulate the orbital cytokine balance away from continued inflammation and antigen presentation and toward disease remission. As such, there may be a role for IL-10 in the treatment of GO, as has been suggested for type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus (10).

IL-8, also called the monocyte-derived neutrophil chemotactic factor, functions primarily as a neutrophil chemoattractant and activating factor (16, 19, 20). This cytokine has been detected using immunohistologic and in situ hybridization assays in virtually every acute inflammatory response. The increased expression of this chemokine in five of six GO patients points to an ongoing inflammatory process in the orbit, even several years following onset of the disease (Fig. 2).

Several studies have shown increased circulating IL-6 in patients with active GO (21, 22, 23). IL-6 is synthesized by a variety of cells including fibroblasts, T cells, macrophages, and endothelial cells in response to IL-1 and, to a lesser degree, TNF. The presence of IL-6 has also been demonstrated in situ in retrobulbar tissues obtained from patients with GO (24) and in vitro using cultured orbital fibroblasts from these patients (25). In addition, we have shown recently that this cytokine is a potent stimulator of TSH receptor expression in cultured GO orbital fibroblasts (26). The current study, showing increased IL-6 in the GO orbit, further supports the potentially important role of this cytokine in GO pathogenesis.

Several groups of investigators used T-cell lines or clones derived from GO orbital connective/adipose tissue to analyze in vitro T-cell phenotypes and cytokine secretion (5, 27, 28, 29). These studies aimed to determine whether cell-mediated immunity, as suggested by a predominance of Th1-type clones producing primarily IFN-, or humoral immunity, as suggested by Th-2 clones secreting IL-4 and IL-5, plays the more important role in GO pathogenesis. Two reports indicated that the majority of Graves’ orbital T-cell lines are CD4+ and that these cells produce primarily Th1-type cytokines. Early histochemical and immunoblotting studies were in agreement with these findings, showing strong IFN- reactivity in frozen GO orbital tissue specimens (2, 3). In contrast, a study by our group (29) and another by Grubeck-Loebenstein et al. (27) reported the establishment of both Th1-type and Th2-type T cell lines from GO orbital tissues. We presented evidence that the predominant type of T cell involved may depend on the duration of the patient’s disease, with Th1-type cells being predominant in early disease and Th2-type cells predominating in disease of more remote onset (>2 yr) (29). In the current study, we were unable to detect either IL-4 or IL-5 in any patient’s orbital tissues, regardless of disease duration. These results support the importance of cell-mediated immunity in GO pathogenesis. The fact that no correlation with disease duration was found may reflect the small number of patients studied or differences in techniques used between the studies.

In other studies more technically similar to the current one, investigators have directly assessed cytokine mRNA in GO orbital tissues, rather than in established cultures of tissue-derived T cells. McLachlan et al. (30) amplified IL-4 or IL-5 in orbital tissue from three of five patients with GO and detected no significant IFN- gene expression in any of the specimens examined. They did not study IL-1, IL-6, and TNF- expression levels and so were unable to comment on the primarily macrophage-derived cytokines. Pappa et al. (5) found both Th-1 and Th-2 cytokine gene expression in extraocular muscle specimens from five patients with GO. A recent study by Hiromatsu et al. (24) examined cytokine gene expression in retroorbital fat and extraocular muscle specimens from patients with GO using RT-PCR. Th1-like cytokine genes (especially IFN-) were predominant in the extraocular muscle tissue of most patients, and the cytokine profile in the orbital fat specimens was quite variable. IL-10 mRNA tended to be predominantly amplified in orbital fat tissue, compared with extraocular muscle tissue.

We are aware of no studies other than the current one that directly compare the expression of various cytokine genes in diseased orbital tissue specimens with expression in normal specimens. We were able to amplify IL-1ß, IL-6, IL-6R, IL-8, IL-10, TNF-, and IFN- genes using mRNA from both normal and GO orbital tissues. By comparing these two groups of specimens, we could determine whether a particular cytokine was truly elevated in the patient samples. In contrast, other studies have interpreted the mere ability to amplify a particular cytokine in disease tissues as suggesting pathologic significance. A limitation inherent in all studies using tissue specimens derives from the fact that tissues are harvested at a particular point in time. As such, a comprehensive overview of pathogenesis cannot be gained from individual specimens. However, these studies do allow for simultaneous comparisons of a number of tissue samples that can be characterized according to pertinent clinical information.

In conclusion, the increased expression of the macrophage-derived cytokines IL-1ß, TNF-, and IL-10 in GO orbital tissues suggests the presence of macrophage activation and ongoing antigen-presentation within the orbit. In addition, the overexpression of IFN-, without evidence of IL-4 or IL-5 expression, supports the concept that cell-mediated, rather than humoral, immunity plays the predominant role in pathogenesis of this disorder.

	Footnotes

 
This work was supported in part by NIH Grant EYO8819 (to R.S.B.) from the National Eye Institute.

Abbreviation: GO, Graves’ ophthalmopathy.

Received March 5, 2003.

Accepted May 20, 2003.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kumar, S. Articles by Bahn, R. S. Search for Related Content PubMed PubMed Citation Articles by Kumar, S. Articles by Bahn, R. S.