Introduction Diabetic retinopathy (DR) is the most common cause of visual loss in the working population and one of the primary reasons for blindness worldwide [ 1, 2]. Diabetic vessel changes may induce ischemia, leading to an upregulation of angiogenic factors in the retina. Aside from the angiogenic component, there is a large body of evidence indicating that inflammation is an important event in the pathogenesis of DR [ 3]. Leukocytes play a major role in the hypoxic retina by producing inflammatory cytokines. Evidence from the retinal ischemia-reperfusion injury model has demonstrated that inflammatory chemokines substantially contribute to inducing retinal damage by infiltration of ocular tissue by leukocytes [ 4]. A recent study investigated vitreous chemokine levels by multiplex bead analysis in 58 patients with vitreoretinal disorders such as uveitis, choroidal neovascularization, proliferative vitreoretinopathy, and proliferative DR (PDR) [ 5]. The present study is the first to examine interferon-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein MIP-1α, macrophage inflammatory protein MIP-1β regulated upon activation, normal T-expressed and secreted (RANTES), and vascular endothelial growth factor (VEGF) in the vitreous and serum by multiplex bead analysis in a large number of diabetic patients compared to controls. Chemokines are small molecular weight proteins that guide the migration of responsive cells. Cells attracted by chemokines follow the direction of increasing chemokine concentration. In addition to chemoattraction, inflammatory chemokines cause activation of leukocytes. Chemokines are categorized into four subgroups: CXC, CC, C, and CX3X [ 6]. IP-10 is a CXC chemokine that contributes to the T-helper type 1immune reactivity. It is secreted by monocytes, endothelial cells, and fibroblasts, and it promotes chemoattraction for monocytes and T-cells [ 7]. Earlier studies described elevated serum IP-10 concentrations as a risk factor for type 1and type 2 diabetes mellitus, and it has been suggested that vitreous levels of IP-10 are increased in patients with PDR [ 8– 10]. The present study is the first to determine IP-10 in the vitreous by Cytometric Bead Array (CBA) technique. MCP-1, MIP-1α, MIP-1β, and RANTES are members of the CC chemokines. MCP-1 recruits immune cells, such as monocytes. It is produced by retinal endothelial cells and has been implicated in leukostasis in the hypoxic retina [ 3, 4]. In animal models raised MCP-1 mRNA levels after retinal hypoxia induction were determined [ 11]. In rats increased expression of IP-10, MCP-1, MIP-1α, and MIP-1β was found in the hypoxic inner retina [ 4]. Previous data presumed MCP-1 as a potential factor in the proliferative phase of DR [ 12]. MIP-1α and MIP-1β are produced by macrophages and activate human granulocytes such as neutrophils, eosinophils, and basophils, which may lead to acute neutrophilic inflammation [ 13]. MIP-1α and MIP-1β activate granulocytes and induce the release of proinflammatory interleukins such as interleukin-1, interleukin-6, and tumor necrosis factor-α [ 14, 15]. MIP-1α mediates the recruitment of monocytes. In a mouse model, MIP-1α has been identified as a potent inducer of retinal neovascularization during postischemic inflammation [ 16]. The retinal ischemia-reperfusion model showed an upregulation of MIP-1β in the retinal vessels [ 4]. We therefore conclude, that MIP-1β may be involved in the inflammatory element of pathogenesis of retinal neovascularization. An upregulation of serum levels of MIP-1α and MIP-1β has been found in type 1 diabetics [ 17]. RANTES, produced by inflammatory cells, retinal endothelial cells, and retinal pigment epithelial cells, attracts T-cells, eosinophils, and basophils. In addition, RANTES activates natural killer cells [ 18, 19]. In tumor model systems, RANTES seems to be a potent angiogenic factor and may be involved in retinal neovascularization in diabetic patients [ 20, 21]. Immunohistochemical investigations of a donor eye with DR showed the presence of RANTES in the diabetic retina compared with the retina of a nondiabetic subject [ 3]. Further, multiplex bead analysis detected RANTES in the vitreous of patients with PDR [ 5]. VEGF plays a major role in ocular neovascularization. VEGF is not only a major mediator of retinal angiogenesis, but also a potent inducer of vasopermeability [ 22]. The expression of VEGF is upregulated in the retinal pigment epithelium, glial cells, and vitreous fibroblasts in diabetic patients [ 23]. CBA was used to determine vitreous and serum levels of IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, and VEGF. The technology has already been used for the measurement of cytokines and growth factors in vitreous, aqueous humor, plasma and serum samples as well as in tissue culture supernatants [ 24– 28]. The determination of cytokines and growth factors in the vitreous by CBA promises to be of substantial benefit for ophthalmologic investigations because numerous parameters can be measured in parallel using a comparatively small sample volume. Furthermore CBA is faster and more cost effective than ELISA technology. For an improved management of DR a better understanding of the pathogenesis is crucial. To elucidate the role of inflammatory and angiogenic factors in the development of DR we measured both vitreous and serum levels of IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, and VEGF in diabetics and normal controls. |
Methods Study population Diabetes was diagnosed according to the 1999 World Health Organization criteria [ 29]. For all probands, the indications for pars plana vitrectomy were diabetic macular edema, epiretinal membranes, and macular holes. In the diabetes group, 10 had no DR, 10 had non-PDR, and 16 had PDR. Exclusion criteria were previous intraocular surgery, earlier intravitreal therapies, photocoagulation in the preceding three months, uveitis, trauma, vitreous hemorrhage, and retinal detachment. Informed consent was obtained from all probands and controls before entering the study.. Research was performed in accordance with the ethical standards of the Declaration of Helsinki [ 30]. Gender and age breakdown Patients were recruited from the Department of Ophthalmology of the Medical University Graz. The population of this case-control study consisted of 36 patients with diabetes mellitus (21 non-insulin dependent, 15 insulin dependent) and 69 non-diabetic control subjects. The mean age of the 36 diabetic subjects (15 men and 21 women) was 66.2 years (SD+-12.2 years). Twenty-one probands were insulin-dependent and 15 probands were non-insulin-dependent. The mean age of the 69 non-diabetic control subjects (23 men and 46 women) was 67.9 years (SD+-10.3 years). With regard to age no significant difference between both groups was observed (p>0.05). The mean hemoglobin A1c (HbA1c) of the diabetic subjects was 7.40 +-1.11% compared to 5.7 +-0.2% in control subjects (normal range HbA1c: 4.0 - 6.0%) (p<0.05). Sample collection and preparation Vitreous Undiluted vitreous samples were obtained during a standard three-port pars plana vitrectomy. After the patient was anesthetized, vitreous fluid (approximately 1 mL) was collected via pars plana using a 20- or 23-gauge vitreous cutter on a 5 mL-syringe after the infusion port was made without running the fluid on. The specimen was immediately transferred to a sterile plastic tube on ice. Promptly the sample was centrifuged at 5000 rpm for 10 min, aliquoted, and stored at –70 °C until assayed. Serum Blood samples were obtained by venous puncture directly before vitrectomy, and immediately placed on ice. Directly each sample was centrifuged at 3000 rpm for 10 min, aliquoted, and stored at –20 °C until assayed. IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, and VEGF were determined in serum as well as in vitreous samples by CBA. Cytometric bead array Concentrations of six different angiogenic factors were determined in both serum and vitreous samples using the BDTM CBA Flex Set System and BDTM Human Soluble Protein Master Buffer Kit (CatNo. 558246, BD Bioscience-PharMingen, San Diego, CA) and the BDTM CBA Flex Sets for the measurement of IP-10 (CatNo. 558280, BD Bioscience-PharMingen), MCP-1 (CatNo. 558287, BD Bioscience-PharMingen), MIP-1α (CatNo 558325, BD Bioscience-PharMingen), MIP-1β (CatNo 558288, BD Bioscience-PharMingen), RANTES (CatNo 558324, BD Bioscience-PharMingen), and VEGF (CatNo. 558336, BD Bioscience-PharMingen) levels. Each BDTM CBA Flex Set contained one bead population with distinct fluorescence intensity as well as the appropriate phycoerythrin (PE) detection reagent and standard. Five bead populations coated with capture antibodies specific to IP-10, MCP-1, MIP-1α, MIP-1β, and VEGF were mixed with each other. The CBA for RANTES was run separately because the serum samples had to be prediluted. RANTES was also determined in serum and vitreous samples by ELISA technique (Quantikine RANTES ELISA, R&D Systems, Minneapolis, MN). The tests were performed according to the manufacturer’s advice, and samples were run in duplicate. As previously described [ 24] the mixed bead populations as well as the single bead population for RANTES were incubated with recombinant standards or test samples to form sandwich complexes. After addition of PE-conjugated detection antibodies, the samples were incubated again and then resolved in the FL-3 channel of a FACSCalibur flow cytometer (BD Bioscience). The results were generated in graphic and tabular format by using the CBA analysis software (BD Bioscience-PharMingen). The assay sensitivities were as follows: IP-10 0.4 pg/mL, MCP-1 1.3 pg/mL, MIP-1α 4.6 pg/mL, MIP-1β 1.4 pg/mL, RANTES 3.2 pg/mL, and VEGF 39.2 pg/mL. For the detection of IP-10, MCP-1, MIP-1α, MIP-1β, and VEGF, serum and vitreous samples were run without predilution. For the detection of RANTES, serum samples were prediluted 1:50, and the vitreous samples were tested undiluted. The tests were performed and analyzed according to the manufacturer’s advice. In brief, 50 µL of the five mixed as well as the single capture beads were mixed with 50 µL of the provided standards or samples (serum, vitreous) and incubated in the dark for 1 h at room temperature. Subsequently 50 µL of the mixed as well as the single PE detection reagent(s) were added, and the samples were incubated in the dark for 2 h. After the second incubation step, the samples were washed, centrifuged (at 200xg for 5 min) and resuspended in 300 µL of wash buffer as previously described [ 24]. The BD FACSCalibur flow cytometer was calibrated with setup beads and 300 events were acquired for each factor and each sample, respectively. Individual analyte concentrations were indicated by their fluorescence intensities (FL-2) and were computed by using the respective standard reference curve and BD CBA software. Statistical analysis Data were analyzed using the statistical package for social sciences (SPSS Version 11.0) for Windows. Group differences between samples from diabetics and controls were analyzed using a Student two-tailed t-test or two-tailed Mann–Whitney test depending on normality assumptions and homogeneity of variances. All tests were performed at an error level of 5%. Due to multiple univariate testing, the Bonferroni correction algorithm of error I level was applied to retain the global error level at 5%. |
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