Abstract
Purpose
Sarcoidosis is a granulomatous inflammatory disease whose cause is unknown. Based on gene expression profiles of peripheral blood and on immunostaining of lymph nodes from sarcoidosis patients, we recently implicated the transcription factor, STAT-1, in the pathogenesis. We explored the potential contribution of STAT-1 in an ocular manifestation of this disease.
Methods
Conjunctival biopsies from four subjects with known sarcoidosis and from eight subjects with conjunctival granulomas presumed from causes other than sarcoidosis were immunostained for STAT1.
Results
STAT-1 was readily detectable in all biopsies from patients with sarcoidosis and in three controls. The activated form of STAT-1, phosphorylated STAT-1, was also more frequently detected in tissue from subjects with sarcoidosis relative to controls.
Conclusions
The activities of STAT-1 in regulating the inflammatory response suggest that it contributes to the conjunctival granulomas characteristic of sarcoidosis, but its expression is not specific for either sarcoidosis or all granulomatous conjunctival disease.
Keywords: conjunctiva, granuloma, sarcoidosis, STAT-1, pSTAT-1
INTRODUCTION
Sarcoidosis is an immune-mediated disease characterized by granuloma formation in a variety of organs including the lung and lymph nodes. Ocular involvement is detectable in about 40% of patients with sarcoidosis. Ophthalmic manifestations include uveitis, dry eye secondary to lacrimal disease, orbital inflammation, optic nerve granuloma and conjunctival granuloma1. The cause of sarcoidosis is unknown, but the immune system is clearly implicated. Some evidence, such as the finding of mycobacterial catalase-peroxidase in granulomatous tissues suggests that an infection might trigger the response2.
In order to clarify the pathogenesis of sarcoidosis, we recently conducted an analysis of mRNA expression in the peripheral blood of patients with this disease3. Although multiple transcripts were differentially expressed among those patients with sarcoidosis compared to controls, the expression of STAT-1 was especially noted. STAT-1 is a transcription factor which regulates many genes implicated in inflammation. Many proteins can induce STAT-1 expression, but a classic regulator of STAT-1 is interferon gamma4, whose expression in turn responds to multiple factors including mycobacterial infections5. The potential contribution of STAT-1 in the pathogenesis of sarcoidosis was reinforced by finding many transcripts under the regulation of STAT-1 also up regulated in the peripheral blood. Finally, immunostaining showed that the expression of the STAT-1 protein and its activated form, phosphorylated STAT-1 (pSTAT-1), was increased in granulomas from lymph nodes of patients with sarcoidosis but not in hyperplastic lymph node. In theory, STAT-1 could represent a novel target for the therapy of sarcoidosis.
In multisystem diseases such as systemic lupus erythematosus, Crohn’s disease, psoriasis, rheumatoid arthritis, or sarcoidosis, the pathogenesis of inflammation in one organ likely differs in some respects from the pathogenesis of inflammation in another organ. Uveitis, for example, may accompany inflammatory bowel disease but the activity of the eye inflammation frequently correlates poorly with the activity of the bowel inflammation6. Tumor necrosis factor inhibitors sometimes control joint inflammation while inducing psoriasiform skin disease7. Accordingly, we sought to determine if STAT-1 could be implicated specifically in the pathogenesis of conjunctival granulomas associated with sarcoidosis.
MATERIALS AND METHODS
Patients and controls
Blocks of formalin-fixed, paraffin-embedded tissue were obtained from the archived samples of the Ophthalmic Pathology Laboratory of the Casey Eye Institute. The diagnosis of all four patients with sarcoidosis had been confirmed by finding non-caseating granuloma on biopsy from a non-ocular tissue site. None of these patients had an infectious explanation for the granuloma outside the eye. The diagnoses for controls along with their ages and genders are shown in Table 1. The OHSU Institutional Review Board has reviewed our study of surgically discarded tissue and considered this use to be exempt from requiring specific subject consent.
Table 1.
STAT-1 Staining in Conjunctival Granulomas
| Patient | Gender | Age (years) |
Diagnosis | STAT-1 | pSTAT-1 |
|---|---|---|---|---|---|
| 1 | M | 61 | Sarcoidosis | Pos | Pos |
| 2 | F | 81 | Sarcoidosis | Pos | Pos |
| 3 | M | 59 | Sarcoidosis | Pos | QNS |
| 4 | M | 54 | Sarcoidosis | Pos | Pos |
| 5 | M | 57 | Actinic degeneration | Pos | Pos |
| 6 | F | 28 | Actinic degeneration | Neg | Neg |
| 7 | M | 69 | Granuloma, unknown cause | Pos | Pos |
| 8 | F | 75 | Actinic degeneration | Neg | Neg |
| 9 | F | 17 | Follicular conjunctivitis | Neg | Neg |
| 10 | M | 73 | Granuloma, unknown cause | Pos | Pos |
| 11 | M | 61 | Foreign body | Neg | Neg |
| 12 | M | 76 | Granuloma, unknown cause | Neg | Neg |
Abbreviations: M = male; F = female; pSTAT-1 = phosphorylated STAT-1; Pos = positive immunostaining; Neg = negative immunostaining; QNS = quantity not sufficient
Immunostaining
Our technique for immunostaining to detect STAT-1 or phosphorylated STAT-1 has been previously described3. Phosphorylated STAT-1 (pSTAT-1) and nonphosphorylated STAT-1 expression was determined by immunohistochemistry on 5 µm sections with purified rabbit polyclonal antibodies detecting either human STAT-1 or pSTAT-1 around the phosphorylation site of tyrosine 701 (GenScript Corp., Piscataway, NJ, USA). Antigen retrieval was achieved by boiling deparaffinized sections in Tris-EDTA buffer (10 mM Tris, 1 mM ethylenediaminetetraacetic acid, pH 9.0) for 10 minutes. Sections were incubated for 1 hour in blocking solution (4.5% goat serum, 0.36% Triton X-100, 0.1% bovine serum albumin). Primary antibodies or control rabbit IgG were then applied overnight diluted 1:80 in blocking solution. After several washes, sections were incubated with pre-absorbed, alkaline phosphatase-conjugated anti-rabbit antibody for 1 hour (1:200, GeneTex Inc., San Antonio, TX, USA). Immunostaining was visualized by incubating with Fast Red reagent (BioGenex, San Ramon, CA, USA) until coloration was apparent (approximately 2 min.). Slides were counterstained with hematoxylin. Immunostaining was interpreted without knowledge of the diagnosis with regard to sarcoidosis.
Statistics
Ages in the sarcoidosis and control groups were compared with a Mann-Whitney Rank Sum Test. The frequencies of positive staining for STAT-1 or pSTAT-1 in the two groups were compared by a one tailed Fisher Exact test (a priori hypothesis that STAT-1 would be increased in association with sarcoidosis).
RESULTS
The subjects, their ages, genders, and diagnoses are shown in Table 1. The subjects with sarcoidosis included 3 males and one female with an average age of 63.8 ± 11.9 (mean ± standard deviation) years. The controls included 5 males and 3 females with an average age of 53.3 ± 24.9 years. Although the controls tended to be somewhat younger than the subjects with sarcoidosis, this difference was not statistically significant (p=0.9). All four of the subjects with sarcoidosis stained positively for STAT-1. One sample was inadequate for the additional staining for pSTAT-1, but of the remaining three tissues, all stained positively for pSTAT-1 (see Figure 1). Expression of STAT-1 was confined to the area of the granulomas and most, if not all, of the granulomas stained positively. Control staining with species-matched, irrelevant polyclonal IgG was negative for all sarcoidosis subjects and controls. The control subjects included three patients with actinic degeneration of the conjunctiva, three with granulomas of unknown cause, one with a foreign body granuloma, and one with a follicular conjunctivitis. Only three of the eight control subjects had positive staining for both STAT-1 and pSTAT-1. Despite the strong trend, the difference for STAT-1 staining between the controls and subjects with sarcoidosis was not statistically significant by Fisher Exact testing (p=0.07). Of note, two of three controls with positive staining had granulomas of uncertain etiology. At least one of the three patients with granulomas of uncertain etiology had additional evaluation for sarcoidosis including a chest computerized tomographic image of the mediastinum. Although no definitely pathological adenopathy was found, he did have evidence for prior granulomatous disease and he did have detectable mediastinal lymph nodes.
Figure 1.
Conjunctival histology with immunostaining for STAT-1 and pSTAT-1. Rows represent patients 1 through 6. Hematoxylin and eosin (H&E) staining of the conjunctiva reveals epithelioid cells and giant cells forming non-caseating granulomas in all six patients. Immunohistochemistry with anti-STAT-1, anti-pSTAT-1, and non-specific control IgG antibody indicate that STAT-1 and pSTAT staining is present in the granulomas of all patients with sarcoidosis (1–4). In patient 3, there was insufficient tissue for adequate interpretation of pSTAT staining so no photograph is included. Patients 5 and 6 both had actinic degeneration. However, only patient 5 displayed positive staining for STAT-1 and pSTAT-1. Original magnification: 400×.
DISCUSSION
Our study indicates that the expression of STAT-1 and its activated form, pSTAT-1, are both consistently increased in conjunctival granulomas from patients with sarcoidosis and that some, but not all, conjunctival granulomas of other cause also express STAT-1. The observations extend our recent report about this transcription factor in the blood and lymph nodes of patients with sarcoidosis. The prior study did not include patients with other granulomatous conditions. This report establishes that STAT-1 expression is not totally specific for sarcoidosis. However, it is possible that some patients with granulomatous inflammation of unknown cause might have sarcoidosis or a limited form of this disease. STAT-1 plays a major role in granuloma formation since mice which lack STAT-1 have an altered ability to form granuloma8. Following mycobacterial challenge, mice that lack STAT-1 form granulomas that do not exhibit necrosis; this is in contrast to what occurs in control mice with wild type STAT-1.
Numerous multisystem diseases show evidence that one organ might be affected by different mediators of inflammation compared to another organ. For example, patients with uveitis treated with a tumor necrosis factor inhibitor such as etanercept can experience relief of joint inflammation while the eye inflammation does not respond9. Orbital inflammation associated with Wegener’s granulomatosis can progress while other aspects of the disease are well controlled. Patients with sarcoidosis often have symptomatic eye disease and asymptomatic pulmonary disease. Accordingly, although we hypothesized that STAT-1 would be expressed in the conjunctival granulomas of patients with sarcoidosis, we might have found that this transcription factor was not contributing to local eye inflammation.
STAT-1 regulates the expression of many transcripts which are implicated in the immune response4. The upregulation of STAT-1 is consistent with the understanding of sarcoidosis such as its presumed infectious origin5. STAT-1’s role could explain why some patients treated with interferons develop granulomatous disease as a complication10. STAT-1 also contributes to the activation of Vitamin D11 which could lead to hypercalcemia, another characteristic association with sarcoidosis. These findings in combination with our previous report showing elevated STAT-1 expression in the blood of patients with sarcoidosis and the data from this report suggest that STAT-1 should be considered as a possible target to treat refractory sarcoidosis.
ACKNOWLEDGEMENTS
Supported by the William and Mary Bauman Foundation, the Stan and Madelle Rosenfeld Family Trust, the William C. Kuzell Foundation, Research to Prevent Blindness, and NIH Grant EY015858
Footnotes
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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