Purpose
To determine the levels of 23 immune mediators in paired aqueous humor (AqH) and serum samples from patients with birdshot chorioretinopathy (BSCR).
Design
Single-centre case-control study.
Methods
A multiplex immunoassay was used to determine the levels of 23 immune mediators (T-cell, proinflammatory, and vascular-active mediators) in paired AqH and serum of 16 BSCR patients. The AqH of 11 age-related cataract controls served as controls.
Results
AqH levels of the T-cell mediators interleukin (IL)-2 ( P = .044) and IL-17 ( P = .039) and proinflammatory mediators IL-1β ( P = .032), IL-6 ( P = .034), and tumor necrosis factor α ( P = .041) were elevated compared with that of age-related cataract controls. The elevated intraocular levels of IL-1β, IL-17, and tumor necrosis factor α in BSCR samples were higher than their concurrent serum levels. A significant positive correlation of intraocular mediators was noted between IL-17 and both IL-2 ( r = 0.744; P < .0001) and IL-23 ( r = 0.921; P < .0001) and between IL-2 and IL-23 ( r = 0.776; P < .0001). AqH levels of vascular-active mediators were not distinct between the groups.
Conclusions
BSCR patients have elevated intraocular levels of proinflammatory and T cell-associated cytokines. Our results suggest the novel pathogenic concept that BSCR is an autoimmune inflammatory disease restricted to the eye and associated with elevated IL-17.
Birdshot chorioretinopathy (BSCR) is a severe and potentially blinding bilateral intraocular disorder characterized by multiple hypopigmented chorioretinal lesions that often leads to atrophy of the retina and optic disc, resulting in a loss of central visual acuity and visual fields. This disease mainly affects middle-aged white persons, and a slight female predominance has been recorded. The most intriguing feature of BSCR is its strong association of more than 90% with the HLA class I A29 allele (HLA-A29), thus representing the strongest link so far known between an HLA antigen and a human disease. Despite the strong HLA association, the pathophysiology of BSCR is not understood. The development of autoimmune inflammation resulting from the presumed molecular mimicry initiated by infections or other damaging factors is the currently favored hypothesis. This hypothesis sees BSCR as an organ-specific ocular autoimmune disease occurring in HLA-A29–positive patients and caused by T-cell reactivity to as yet unknown antigens or a currently unidentified anomaly of the HLA-A29 molecule. T helper 17 (Th17) cells are a subset of CD4+ T helper cells that derive their name from the characteristic production of interleukin (IL)-17. Th17 responses play an important role in many well-known autoimmune diseases, including rheumatoid arthritis and Crohn disease. Th17 activation also has been reported for HLA-B27–associated spondylitis, another HLA class I-associated disease, and is associated with noninfectious uveitis. To understand better the role of T cells in BSCR, we investigated the profile of immune mediators in paired intraocular fluid and serum samples from BSCR patients. Our report focuses on the elevated intraocular levels of proinflammatory cytokines and IL-17 in BSCR.
Methods
Subjects
This was a transsectional case-control study. Our study cohort included 16 patients with BSCR diagnosed on the basis of research criteria established by an international consensus conference. All patients were HLA-A29 positive and had bilateral chronic uveitis with the characteristic hypopigmented chorioretinal lesions and associated vitritis. We included 11 patients with age-related cataract who had no history of intraocular inflammation or vitreoretinal disease to serve as controls. In this study, we analyzed paired aqueous humor (AqH) and serum samples from patients with BSCR and AqH samples from patients with age-related cataract. AqH and serum samples from patients with BSCR were derived from the remainders of diagnostic samples collected during an active state of disease; samples from patients with cataract were collected during cataract surgery. Undiluted paired AqH and serum samples were stored at −80 C and thawed immediately before analysis.
Multiplex Immunoassay
Undiluted AqH and serum samples were analyzed to isolate 23 soluble mediators by multiplex immunoassay as already described by de Jager and associates. In short, interfering immunoglobulins were removed by protein-L–coated beads. Samples were run undiluted and diluted 1/50 in HighPerformance enzyme-linked immunosorbent assay buffer (HPE-buffer; Sanquin, Amsterdam, The Netherlands). All assays were carried out directly in a 96-well 1.2-Am filter plate (Millipore, Amsterdam, The Netherlands) at room temperature. In a volume of 10 μL/well, each mediator (1000 microspheres) was incubated with standard, sample, or blank. An equal volume (10 μL) of biotinylated antibodies (16.5 μg/mL) was added to the wells and incubated for 60 minutes. Next, beads were washed (phosphate-buffered saline, 1%; bovine serum albumin, 0.5%, Tween 20; pH, 7.4) and incubated for 10 minutes with (50 ng/well) streptavidin R-phycoerythrin (BD Biosciences, San Diego, California, USA). Beads were washed twice and brought in 100 μL HPE-buffer to measure fluorescence intensity using a Bio-Plex system in combination with the Bio-Plex Manager software version 3.0 using 5 parametric curve fitting (Bio-Rad Laboratories, Hercules, California, USA). The mediators were categorized as T-cell mediators (IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, IL-17, IL-18, IL-22, IL-23, interferon γ), proinflammatory mediators (IL-receptor antagonist, IL-1β, IL-6, IL-8, interferon-inducible protein of 10 kDa, tumor necrosis factor [TNF] α, monocyte-chemotactic protein 1, macrophage-inhibitory protein 1 α, macrophage-derived chemokine 1), and vascular-active mediators (soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, and vascular endothelium growth factor).
Statistical Analysis
Statistical analysis was performed using Graphpad Prism software version 5.0 (Graphpad Software, Inc, La Jolla, California, USA) and SPSS software version 15.0.1 (SPSS, Inc, Chicago, Illinois, USA). Group differences were analyzed using the Mann–Whitney U test with 2-sided P values with Bonferroni correction for multiple comparisons (α = 0.05/23 = 0.0022). A P value of less than .05 was regarded as statistically significant. Correlations between mediator concentrations were analyzed according to the Pearson product-moment correlation coefficient using the rank test. Mediator concentrations more or less than the detection limit were given as the highest or lowest detectable value. Statistical analysis concentrations less than the detection limit were converted to a value of 0.5 times the lowest point of the calibration curve. If the concentrations of the raw data were more than the maximal levels, they were set to the maximal level of each standard curve.
Results
The mean age ± standard deviation of the BSCR patients was 61 ± 13 years, ranging from 27 to 84 years. The mean age ± standard deviation of age-related cataract patients was 73 ± 12 years, ranging from 50 to 92 years ( Table ). BSCR patients were found to have elevated intraocular levels of T-cell mediators; more specifically, intraocular levels of IL-17 were higher in BSCR patients than in controls ( P = .039). The AqH and serum levels of the 23 mediators in BSCR patients are given in the Table . BSCR was associated further with elevated intraocular IL-2 levels ( P = .044). In addition, BSCR patients had increased intraocular levels of the proinflammatory mediators IL-1β ( P = .032), IL-6 ( P = .034), and TNF-α ( P = .041). The elevated intraocular levels of IL-1β, IL-17, and TNF-α in BSCR patients were higher than their concurrent serum levels ( Table ). IL-22 could not be detected in either the AqH or serum. AqH levels of vascular-active mediators were similar in both groups.
| Birdshot Chorioretinopathy | Age-Related Cataract | ||||
|---|---|---|---|---|---|
| No. | 16 | 11 | |||
| Mean age ± standard deviation (yrs) | 61 ± 13 | 73 ± 12 | |||
| Male-to-female ratio | 1:15 | 6:5 | |||
| Mediator | AqH | Serum | AqH | P Value | |
|---|---|---|---|---|---|
| BSCR AqH vs Cataract AqH | BSCR AqH vs BSCR Serum | ||||
| IL-1RA | |||||
| Geometric mean | 51.6 | 58.8 | 89.98 | — | — |
| Range | 10.8 to 178.7 | 2.4 to 387.1 | 10.8 to 998.6 | ||
| IL-1β | |||||
| Geometric mean | 3.9 | 1.8 | 2.4 | .032 | .005 |
| Range | 1.9 to 10 | < 1.5 to 6.2 | 1.5 to 3.1 | ||
| IL-2 | |||||
| Geometric mean | 10.5 | 12.3 | 4.2 | .044 | — |
| Range | 1.8 to 25.3 | 1.8 to 990.8 | 1.0 to 12.9 | ||
| IL-4 | |||||
| Geometric mean | 13.5 | 3.1 | 10.1 | — | <.0001 |
| Range | 7.6 to 21.8 | 2.4 to 37.8 | 7.5 to 16.3 | ||
| IL-5 | |||||
| Geometric mean | 5.3 | < 1.2 | 4.4 | — | — |
| Range | 3.2 to 9.3 | < 1.2 | 1.6 to 6.2 | ||
| IL-6 | |||||
| Geometric mean | 16 | 2.9 | 5.7 | .034 | — |
| Range | 5.7 to 297.9 | < 2.4 to 28.6 | 2.4 to 14.6 | ||
| IL-8 | |||||
| Geometric mean | 34.5 | 26.6 | 38.2 | — | — |
| Range | 13.8 to 310.9 | < 5.3 to 870.5 | 9.5 to 119.1 | ||
| IL-10 | |||||
| Geometric mean | 2.9 | < 2.3 | < 2.3 | — | — |
| Range | < 2.3 to 10.7 | < 2.3 to 11.8 | < 2.3 to 1.9 | ||
| IL-12p70 | |||||
| Geometric mean | 54.7 | 30.5 | 45.4 | — | — |
| Range | 36.5 to 94.8 | < 4.3 to 1454 | 35.1 to 77.8 | ||
| IL-13 | |||||
| Geometric mean | 18.1 | 2.2 | 16.1 | — | .005 |
| Range | 6.8 to 34.5 | < 1.1 to 135.6 | 10.5 to 23.6 | ||
| IL-17 | |||||
| Geometric mean | 243.7 | < 1.1 | 142.8 | .039 | <.0001 |
| Range | 158.8 to 462.5 | < 1.1 to 10 | 25.2 to 232.7 | ||
| IL-18 | |||||
| Geometric mean | 28.9 | 111.9 | 42.2 | — | .012 |
| Range | 0 to 71.1 | 12.2 to 387 | 23.1 to 61.8 | ||
| IL-23 | |||||
| Geometric mean | 6.8 | 1.7 | 5.1 | — | — |
| Range | 4.5 to 13.3 | 1.2 to 507.1 | 3.2 to 8.2 | ||
| TNF-α | |||||
| Geometric mean | 2 | 1.8 | < 1.2 | .041 | .012 |
| Range | < 1.2 to 4.8 | < 1.2 to 38.2 | < 1.2 to 1.9 | ||
| IFN-γ | |||||
| Geometric mean | < 9.1 | < 9.1 | < 9.1 | — | — |
| Range | < 9.1 to 10.3 | < 9.1 to 74.4 | < 9.1 | ||
| CCL2/MCP-1 | |||||
| Geometric mean | 662 | 106 | 459.6 | — | <.0001 |
| Range | 247.3 to 2020 | 21.2 to 670.9 | 195.6 to 1738 | ||
| CCL3/MIP-1α | |||||
| Geometric mean | 26.4 | 9.3 | 15.2 | — | — |
| Range | 15.2 to 48.4 | < 9.3 to 90 | < 9.3 to 26.4 | ||
| CCL22/MDC | |||||
| Geometric mean | 20.4 | 320.3 | 32.7 | — | <.0001 |
| Range | 8.8 to 55.5 | 106.5 to 1146 | 10.8 to 357.4 | ||
| CXCL10/IP-10 | |||||
| Geometric mean | 945.3 | 107.2 | 242.3 | — | <.0001 |
| Range | 110.3 to > 8530 | 20.3 to 788.5 | 37.6 to 1601 | ||
| sICAM-1 | |||||
| Geometric mean | 326.7 | 550.1 | 254.6 | — | <.0001 |
| Range | 119.2 to 1030 | 339.9 to 650.7 | 94.2 to 610.8 | ||
| sVCAM-1 | |||||
| Geometric mean | 3408 | 7403 | 3318 | — | .025 |
| Range | 818.3 to 12897 | 26.6 to 20935 | 175.1 to 6512 | ||
| VEGF | |||||
| Geometric mean | 156.4 | 150.4 | 155.3 | — | — |
| Range | 16.7 to 531.2 | 45.3 to 760.2 | 62.9 to 277.9 | ||
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