Purpose
To determine a possible implication of CD21, CD35, and CD55 in the pathogenesis of age-related macular degeneration (AMD) by assessing the difference in expression rates of these factors on AMD patients and a control group.
Design
Case-control study.
Methods
Fifty unrelated AMD patients and 48 unrelated sex- and age-matched control subjects participated in this case-control study. Samples of fresh EDTA-blood were stained and flow cytometry was chosen to measure fluorescence emissions. The association between exudative AMD and CD21, CD35, and CD55 was evaluated from all patients who completed the study.
Results
Our study shows CD35 to be expressed in a significantly higher frequency in AMD patients on monocytes ( P = .00586), lymphocytes ( P = .000605), and granulocytes ( P < .000033). In contrast, the expression rate of CD21 ( P > .05) and CD55 ( P > .05) are similar in both groups.
Conclusion
More regulative factors of the complement system are involved in pathogenesis of AMD. Our study underlines the key role of the complement system in AMD and shows the involvement of the whole immune system through more regulative factors.
Age-related macular degeneration (AMD) is a progressive degenerative disorder in the central region of the retina and the leading cause of legal blindness in people aged over 55 years in developed countries. In the pathogenesis of this disease, the complement factor H (CFH) polymorphism Y402H in AMD strongly suggests to play an important role in the complement system in certain ethnic groups. The complement system is part of the innate immune system and is closely associated with the cellular response and the adaptive immune system.
Based on similarities between complement factor H and the decay-accelerating factor (CD55), complement receptor 2 (CD21), and complement receptor 1 (CD35) concerning structural organization, gene locus, and inhibitory influence on the complement system, these 3 factors were chosen to determine a possible implication in the pathogenesis of age-related macular degeneration. All these factors are involved in regulation of the activated complement cascade. Taking the hypothesis of complement-mediated AMD into account, a significant variation of the expression of these molecules in patients with AMD might be expected.
The aim of this study is to assess a difference in expression rates of CD21, CD35, and CD55 on AMD patients and a healthy control group by analyzing the mean channel fluorescence (MCF) with flow cytometry (fluorescence-activated cell sorting [FACS]).
Methods
Ninety-eight consecutive patients consulting our department of ophthalmology were included in this case-control study over a 3-month period: 50 unrelated patients with exudative AMD and 48 unrelated age- and sex-matched control subjects. All patients were aged 55 or older, were of caucasian origin, and lived in the same geographic area of Austria.
According to the Age-Related Eye Disease Study system for classification of AMD, all AMD patients enrolled in the present study were classified with exudative age-related macular degeneration according to the angiographic findings (including predominantly classic choroidal neovascularization [CNV], minimal classic choroidal neovascularization, occult choroidal neovascularization, and retinal angiomatous proliferation). Exudative AMD was diagnosed by ophthalmoscopic fundus examination, optical coherence tomography, and fluorescein/indocyanine angiography. Excluded from the study were patients with hereditary diseases, polypoidal choroidal vasculopathy, or secondary CNV attributable to pathologic myopia (greater than negative 2 diopters), angioid streaks, inflammatory, infectious chorioretinal disease, trauma, diabetic retinopathy, general acute inflammatory or infectious disease.
Selection of the control group and matching was performed by sex and age to within 5 years in order to provide a control group with similar distribution of the matching variables. The control group, clinic patients with a non-AMD diagnosis (for example, symptoms of conjunctivitis but negative swab, small conjunctival foreign bodies), had a thorough eye examination with a detailed fundus examination. Exclusion criteria for controls were evidence of any stage of age-related maculopathy, macular hemorrhages of any cause, or media opacities resulting in impaired visualization of the macula—cataract grade 3 and 4 according to the Lens Opacities Classification System III (LOCS system). Controls having an acute or chronic inflammatory disease or infectious disease were also excluded.
Patients and controls with autoimmune diseases, collagen diseases, or renal failure, as well as rheumatoid arthritis, osteoarthritis, chronic inflammatory lung disease, inflammatory bowel disease and psoriasis were excluded.
An aliquot of 5 mL of venous blood from each subject was withdrawn and collected in an EDTA-containing tube. As suggested by the company Becton Dickinson (San José, California, USA), the blood samples were used within 6 hours for optimal results and stored at room temperature (20 to 25 C) until staining. Company instructions were followed. After staining of the samples a maximum of 1 hour of storage is recommended at cool temperatures (2 to 8 C).
Staining of the Erythrocytes
Staining of erythrocytes was performed according to standard protocols. In brief, whole blood was diluted 10-fold with CellWash (Becton Dickinson [BD] Biosciences) and 50 μL of this suspension was used for staining with 10 μL antibody γ1/γ2a isotype control (FITC-marked IgG1 and PE-marked IgG2a) or with 10 μL mouse anti-human CD55 (FITC-marked/Southern Biotech).
Staining of the Leukocytes
Staining of leukocytes with anti-human CD21-PE antibody and anti-human CD35-FITC antibody was performed in 100 μL of EDTA–whole blood according to standard protocols (Becton Dickinson). Erythrocytes were lysed with BD lysis solution for 10 minutes at room temperature.
Flow Cytometric Analyses
Flow cytometric analyses were performed on a FACScan flow cytometer (Becton Dickinson) equipped with an argon-ion laser tuned at 488 nm. Data acquisition and analysis were performed using CellQuest Version 3.1f (Becton Dickinson [BD] Immunocytometry Systems). A total of 1000 cells were analyzed from each sample. For each single experiment on the flow cytometry the instrument was calibrated with FITC- and PE-labeled beads to generate a standard calibration curve. Granulocytes, monocytes, and lymphocytes were differentiated and tested from the leukocyte staining.
Statistics
A sample size calculation was performed using an alpha error of 0.05 (5%), a beta error of 0.20 (20%), and a clinically significant difference of half of the standard deviation of CD35 according to the literature to determine the power calculation.
Statistical analysis was performed with SPSS for Windows (version 6.0; IBM, Somers, New York, USA). Results are expressed in mean ± standard deviation (SD). The comparison between groups was assessed by 2-tailed Student test. All P values smaller than .01 were considered as statistically significant. The critical boundary of .01 results from correction for multiplicity according to Bonferroni due to the number of tests (5 tests were performed, .05/5 = .01).
Results
Fifty patients with exudative AMD were enrolled in this study and classified into subgroups according to angiographic findings. Forty-eight unrelated controls were age- and gender-matched. The AMD group consisted of 30 women and 20 men, and the mean age was 78.0 (± 7.1 SD) years. The control group consisted of 26 women and 22 men, and the mean age was 76.5 (± 6.5 SD) years.
Higher expression of CD35 in AMD patients was detected, showing different significant results on granulocytes, monocytes, and lymphocytes. The CD35 expression on granulocytes shows significant results with P = .000033: AMD patients showed elevated MCF compared to the control group: 109.83 MCF (± 46.07 SD; median 112.69, range 205.95) on AMD patients vs 73.66 MCF (± 34.21 SD; median 71.01, range 137.63) on the controls.
The CD35 expression on monocytes showed a higher expression in AMD patients compared to the healthy controls ( P = .00586): 62.24 MCF (± 30.32 SD; median 56.69, range 188.58) vs 46.63 MCF (± 23.30 SD; median 46.10, range 110.59). The CD35 expression on lymphocytes also showed a higher expression in AMD patients compared to the healthy controls ( P = .000605): 3.87 MCF (± 1.16 SD; median 3.48, range: 7.24) vs 3.06 MCF (± 0.59 SD; median 2.98, range 3.64).
In contrast, CD21 on B cells and CD55 on erythrocytes showed similar expression rates. The CD55 expression on erythrocytes showed following results: 19.50 MCF (± 3.44 SD; median 19.42, range 18.56) in AMD patients vs 18.91 MCF (± 3.38 SD; median 19.23, range 14.23) in the control group ( P > .05).
The CD21 expression in AMD patients compared to the controls showed the following values: 40.57 MCF (± 11.94 SD; median 39.73, range: 61.54) vs 39.97 MCF (± 14.57 SD; median 40.17, range 71.59) ( P > .05).
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