Mads Krüger Falk

Altered Expression of CD46 and CD59 on Leukocytes in Neovascular Age-Related Macular Degeneration

PURPOSE To investigate the expression of the complement regulatory proteins CD46, CD55, and CD59 on peripheral leukocytes in neovascular age-related macular degeneration (AMD). DESIGN Prospective, case-control study. METHODS Thirty-five unrelated patients with neovascular AMD and 30 control individuals were included in this case-control study. All participants were subjected to a structured interview and detailed imaging (autofluorescence, digital funduscopy, spectral-domain optical coherence tomography, and fluorescein and indocyanine green angiography in patients suspected of having neovascular AMD) was performed. Fresh ethylenediamine-tetraacetic acid blood was obtained and stained with monoclonal antibodies. Using flow cytometry, the percentage of CD14(+) monocytes, CD45(+) lymphocytes, and CD45(+) granulocytes positive for CD46, CD55, and CD59 was determined in patients with neovascular AMD and was compared with that of controls. RESULTS We found that the expression of CD46 and CD59 was significantly lower on CD14(+) monocytes in patients with neovascular AMD compared with controls (P = .0070). A significantly lower expression of CD46 on lymphocytes was observed in patients with fibrosis compared with patients without fibrosis (P = .010). CONCLUSIONS Our study suggests that neovascular AMD is associated with an inadequate regulation of the complement system, supporting current evidence on the role of complement dysregulation in the pathogenesis of AMD.

Age-related Macular Degeneration Is Associated with Increased Proportion of CD56+ T Cells in Peripheral Blood

PURPOSE To examine the association between age-related changes in the T-cell compartment and prevalence of age-related macular degeneration (AMD). DESIGN Case-control study. PARTICIPANTS A total of 117 AMD cases and 106 controls were included prospectively. METHODS Fresh-drawn peripheral blood samples were processed for flow cytometric analysis of T-cell populations. Plasma samples were analyzed for anti-cytomegalovirus (CMV) immunoglobulin (Ig)G and complement factor H (CFH) Y402H genotype. The diagnosis of AMD was made according to the Clinical Age-Related Maculopathy Staging System. MAIN OUTCOME MEASURES Association between frequency of aged T cells and prevalence of AMD. RESULTS The prevalence of AMD was associated with distinct age-related changes in the T-cell compartment. Specifically, the patients with AMD had an increased frequency of CD28(-) T cells that expressed the CD56 surface marker (patients, 34.9% vs. aged controls, 25.8%; P = 0.002). Participants in the highest tertile of CD56(+) CD28(-) T cells had an odds ratio (OR) for the presence of AMD of 3.2 (95% confidence interval [CI], 1.2-8.8) after adjustment for CFH genotype, anti-CMV IgG positivity, age, sex, and smoking history. The adjusted OR of the presence of AMD for persons having at least 1 CFH H402 risk allele increased from 3.5 (95% CI, 1.5-8.1) to 13.3 (95% CI, 3.3-53.6) for persons with at least 1 CFH H402 risk allele and above the median level of CD56(+) CD28(-) T cells. CONCLUSIONS We found increased levels of circulating aged CD56(+) CD28(-) T cells in patients with AMD. Although this supports the notion of AMD as a systemic disease, it also suggests that the adaptive immune system is implicated in its pathogenesis.

Increased Expression of CD200 on Circulating CD11b+ Monocytes in Patients with Neovascular Age-related Macular Degeneration

OBJECTIVE Dysregulation of retinal microglial activity has been implicated in the pathogenesis of neovascular age-related macular degeneration. Microglia activity can be regulated through the membrane protein CD200 and its corresponding receptor, the CD200 receptor (CD200R). Because both the ligand and the receptor are expressed on a broad spectrum of cell types, we set out to study the expression of CD200 and CD200R on CD11b+ monocytes, granulocytes, and subsets of T lymphocytes. DESIGN Prospective, case-control study. PARTICIPANTS The study population consisted of 62 patients with neovascular age-related macular degeneration (AMD) and 44 age-matched controls without AMD. METHODS The participants were aged 60 years or older, had no history of immune dysfunction or cancer, and were not receiving immune-modulating therapy. All participants were subjected to a structured interview, and detailed retinal imaging was performed: fundus autofluorescence imaging, digital color fundoscopy, and spectral-domain optical coherence tomography. Fluorescein and indocyanine green angiography were performed in patients with suspected neovascular AMD. Visual acuity was measured in both eyes. Fresh venous blood was obtained and stained with monoclonal antibodies and analyzed using flow cytometry within 6 hours of phlebotomy. MAIN OUTCOME MEASURES The percentage of CD11b+ monocytes, granulocytes, and CD4+/CD8+ T lymphocytes positive for CD200 or CD200R in patients and controls, respectively. RESULTS Patients with neovascular AMD had a higher percentage of CD11b+CD200+ monocytes and CD200+ monocytes compared with controls. Multiple regression analysis revealed that the intergroup differences observed were independent of age. Moreover, an age-related increment in CD200 expression on monocytes was observed in controls with healthy eyes, but not in patients with neovascular AMD. We did not find any differences in CD200 and CD200R expression between patients with subretinal fibrosis and patients without subretinal fibrosis. CONCLUSIONS The surface expression of CD200 on circulating CD11b+ monocytes was found to be increased in patients with neovascular AMD compared with controls with healthy eyes. This novel finding supports the notion that altered regulation of the inflammatory response plays an integral role in the pathogenesis of AMD. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

The Association between Plasma 25-Hydroxyvitamin D and Subgroups in Age-Related Macular Degeneration: A Cross-Sectional Study

Objectives To evaluate potential differences in plasma 25-hydroxyvitamin in subtypes of age-related macular degeneration (AMD), and in patients in Clinical Age-Related Maculopathy Staging (CARMS) group 5 with or without subretinal fibrosis. Methods This single-center cross-sectional study included 178 participants during a period of 20 months. Ninety-five patients belonged to CARMS 5; twelve belonged to CARMS 4; twenty-two belonged to CARMS 2 or 3; and 49 individuals did not have AMD (CARMS 1). Following a structured interview, a detailed bilateral retinal examination was performed and participants were allocated to their respective subgroups in accordance with the Clinical Age-Related Maculopathy Staging system. Plasma 25-hydroxyvitamin D2 and D3 were analyzed using liquid chromatography-tandem mass spectrometry. Genomic DNA was extracted from leukocytes and genotyped for single nucleotide polymorphisms (SNPs) in the vitamin D metabolism. Differences in plasma 25-hydroxyvitamin D were determined in the subgroups as well as between patients in CARMS 5 with or without subretinal fibrosis. Results Plasma 25-hydroxyvitamin D was comparable in patients across CARMS groups 1 to 5 (p = 0.83). In CARMS 5, the presence of subretinal fibrosis was associated with significantly lower concentrations of 25-hydroxyvitamin D as compared to the absence of subretinal fibrosis (47.2 versus 75.6 nmol/L, p<0.001). Patients in CARMS 5 with subretinal fibrosis were more likely to have insufficient levels of 25-hydroxyvitamin D compared to patients without subretinal fibrosis (p = 0.006). No association was found between the SNPs rs10877012, rs2228570, rs4588, or rs7041 and AMD subgroups or plasma 25-hydroxyvitamin levels. Conclusions This study suggests that the presence of subretinal fibrosis in patients belonging to CARMS 5 may be associated with a poor vitamin D status. Our observations warrant further investigation into the role of vitamin D in the development of subretinal fibrosis.

In patients with neovascular age-related macular degeneration, physical activity may influence C-reactive protein levels

Purpose Association of neovascular age-related macular degeneration (AMD) with C-reactive protein (CRP) was previously reported, indicating a relation to systemic low-grade inflammation. However, visual impairment limits physical activity, and physical activity modulates CRP levels. Here, we investigated the impact of physical activity on CRP levels in patients with neovascular AMD and control individuals. Subjects and methods We recruited participants from our outpatient AMD program, and control individuals from non-AMD patients, visitors, and department staff. After initial screening of 191 individuals, we included 98 patients with neovascular AMD and 77 controls. All were screened using digital fundus photography and optical coherence tomography, and interviewed about medical history and physical activity. Venous blood samples were obtained for high-sensitivity CRP. Results Physically active individuals had lower CRP than physically inactive individuals (P=0.003), and physical activity was associated with lower CRP in patients (P=0.038) and controls (P=0.031). Patients and controls did not differ in percentage physically active (P=0.807) or in overall CRP levels (P=0.394). The independent contribution of physical activity on CRP was confirmed in a multiple regression analysis (P=0.009), in which the presence of neovascular AMD did not contribute significantly (P=0.913). Conclusion Our findings suggest that elevated CRP levels in patients with neovascular AMD are at least partly explained by physical inactivity. Future studies of systemic inflammation among the visually impaired should include disease-related implications, such as the impact of physical activity.

Early and exudative age-related macular degeneration is associated with increased plasma levels of soluble TNF receptor II

We have recently identified homeostatic alterations in the circulating T cells of patients with age‐related macular degeneration (AMD). In cultures of retinal pigment epithelial cells, we have demonstrated that T‐cell‐derived cytokines induced the upregulation of complement, chemokines and other proteins implicated in AMD pathogenesis. The purpose of this study was to test whether increased plasma levels of cytokines were present in patients with AMD.

Dysregulation of CXCR3 expression on peripheral blood leukocytes in patients with neovascular age-related macular degeneration.

PURPOSE The chemokine receptor CXCR3 has been strongly related to inhibition of angiogenesis. The purpose of this study was to investigate the association between expression of CXCR3 on peripheral blood leukocytes and age-related wet macular degeneration. Furthermore, we measured the plasma concentration of chemokines CXCL9 to -11. METHODS The study group consisted of patients with age-related macular degeneration (AMD) attending our department. Patients referred for reasons other than AMD were enrolled as control subjects. The expression of CXCR3 on T cells and the plasma concentration of CXCL9 to -11 were measured using flow cytometry. RESULTS We looked at all CD8(+) T cells expressing CXCR3 and found a significantly lower percentage of these cells in the neovascular AMD group compared to the age-matched control group (P = 0.05). When dividing the CD8(+) cells into functional groups according to their expression of CXCR3, we found a significantly lower percentage of CD8(+) CXCR3(high) cells in the group with neovascular AMD compared to the control group (P = 0.038). We found a lower percentage of CD4(+)CD69(+)CXCR3(+) T cells in the group of patients with neovascular AMD when compared to the age-matched control group (P = 0.052). CONCLUSIONS Our results point toward a systemic dysregulation of CXCR3 in patients with neovascular AMD. Since there is evidence to suggest that CXCR3 is able to alter the response of VEGF, the primary driver of choroidal neovascularization (CNV) formation, low levels of CXCR3 could potentially drive some patients toward a more angiogenic profile leading to CNV formation and growth. CXCR3-enhancing molecules could therefore be a possible target for treatment of AMD.

Blood expression levels of chemokine receptor CCR3 and chemokine CCL11 in age-related macular degeneration: a case–control study

BackgroundDysregulation of the CCR3/CCL11 pathway has been implicated in the pathogenesis of choroidal neovascularisation, a common feature of late age-related macular degeneration (AMD). The aim of this study was to investigate the expression of CCR3 and its ligand CCL11 in peripheral blood in patients with neovascular AMD.MethodsPatients with neovascular AMD and healthy controls were included. Blood samples were obtained and prepared for flow cytometry to investigate the expression of CCR3. Levels of CCL11 were measured in plasma using Cytometric Bead Array. Differences between the groups were tested using Kruskal-Wallis test and Mann–Whitney U test.ResultsPatients (n = 83) with neovascular AMD and healthy control persons (n = 114) were included in the study. No significant difference in the expression of CCR3 was found on CD9+ granulocytes when comparing patients suffering from neovascular AMD with any of the control groups. We did not find any alteration in CCL11 levels in patients among the age matched groups. There was no correlation between expression of CCR3/CCL11 and clinical response to treatment with anti-vascular endothelial growth factor (VEGF).ConclusionOur results do not suggest a systemic alteration of the CCR3/CCL11 receptor/ligand complex in patients with neovascular AMD.

Systemic and Ocular Long Pentraxin 3 in Patients with Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) has been associated with both systemic and ocular alterations of the immune system. In particular dysfunction of complement factor H (CFH), a soluble regulator of the alternative pathway of the complement system, has been implicated in AMD pathogenesis. One of the ligands for CFH is long pentraxin 3 (PTX3), which is produced locally in the retinal pigment epithelium (RPE). To test the hypothesis that PTX3 is relevant to retinal immunohomeostasis and may be associated with AMD pathogenesis, we measured plasma PTX3 protein concentration and analyzed the RPE/choroid PTX3 gene expression in patients with AMD. To measure the ability of RPE cells to secrete PTX3 in vitro, polarized ARPE-19 cells were treated with activated T cells or cytokines (interferon (IFN)-gamma and/or tumor necrosis factor (TNF)-alpha) from the basolateral side; then PTX3 protein concentration in supernatants and PTX3 gene expression in tissue lysates were quantified. Plasma levels of PTX3 were generally low and did not significantly differ between patients and controls (P=0.307). No statistically significant difference was observed between dry and exudative AMD nor was there any correlation with hsCRP or CFH genotype. The gene expression of PTX3 increased in RPE/choroid with age (P=0.0098 macular; P=0.003 extramacular), but did not differ between aged controls and AMD patients. In vitro, ARPE-19 cells increased expression of the PTX3 gene as well PTX3 apical secretions after stimulation with TNF-alpha or activated T cells (P<0.01). These findings indicate that PTX3 expressed in the eye cannot be detected systemically and systemic PTX3 may have little or no impact on disease progression, but our findings do not exclude that locally produced PTX3 produced in the posterior segment of the eye may be part of the AMD immunopathogenesis.

CX3CL1/CX3CR1 and CCL2/CCR2 Chemokine/Chemokine Receptor Complex in Patients with AMD

Purpose The chemokine receptors CX3CR1 and CCR2 have been implicated in the development of age-related macular degeneration (AMD). The evidence is mainly derived from experimental cell studies and murine models of AMD. The purpose of this study was to investigate the association between expression of CX3CR1 and CCR2 on different leukocyte subsets and AMD. Furthermore we measured the plasma levels of ligands CX3CL1 and CCL2. Methods Patients attending our department were asked to participate in the study. The diagnosis of AMD was based on clinical examination and multimodal imaging techniques. Chemokine plasma level and chemokine receptor expression were measured by flow-cytometry. Results A total of 150 participants were included. We found a significantly lower expression of CX3CR1 on CD8+ T cells in the neovascular AMD group compared to the control group (p = 0.04). We found a significant positive correlation between CCR2 and CX3CR1 expression on CD8+ cells (r = 0.727, p = 0.0001). We found no difference in plasma levels of CX3CL1 and CCL2 among the groups. Conclusions Our results show a down regulation of CX3CR1 on CD8+ cells; this correlated to a low expression of CCR2 on CD8+ cells. Further studies are needed to elucidate the possible role of this cell type in AMD development.