interleukin-8 and Macular-Degeneration

The results of different studies have indicated the possible associations of TLR4 and IL-8 genes polymorphisms with Age-related Macular Degeneration (AMD). A meta-analysis study was designed to evaluate the possible associations of TLR4 (rs4986790/c.896A>G and rs4986791/ c.1196 C > T) and IL-8 (rs4073/c.251A>T and rs2227306/c.781 C > T) genes polymorphisms with AMD.. A systematic literature search was carried out in PubMed, Embase, Web of Science, and Scopus databases to identify relevant publications. Pooled Odds Ratio (OR) with 95% Confidence Interval (CI) was used to evaluate the power of association.. A total of 12 case-control studies with 4804 AMD patients and 4422 healthy controls were included in this meta-analysis. The analysis of genotypic and allelic models demonstrated significant associations between IL-8 c.781 C > T (CC vs. TT+TC: OR = 0.62 [0.48-0.81],. The current meta-analysis study suggested that IL-8 c.781 C > T polymorphism is associated with susceptibility to AMD.

Topics: Case-Control Studies; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Interleukin-8; Macular Degeneration; Odds Ratio; Polymorphism, Single Nucleotide; Risk Factors; Toll-Like Receptor 4

During age-related macular degeneration (AMD), chronic inflammatory processes, possibly fueled by high glucose levels, cause a breakdown of the retinal pigment epithelium (RPE), leading to vision loss. Phloretin, a natural dihydroxychalcone found in apples, targets several anti-inflammatory signaling pathways and effectively inhibits transporter-mediated glucose uptake. It could potentially prevent inflammation and cell death of RPE cells through either direct regulation of inflammatory signaling pathways or through amelioration of high glucose levels. To test this hypothesis, ARPE-19 cells were incubated with or without phloretin for 1 h before exposure to lipopolysaccharide (LPS). Cell viability and the release of pro-inflammatory cytokines interleukin 6 (IL-6), IL-8 and vascular endothelial growth factor (VEGF) were measured. Glucose uptake was studied using isotope uptake studies. The nuclear levels of nuclear factor erythroid 2-related factor 2 (Nrf2) were determined alongside the phosphorylation levels of mitogen-activated protein kinases. Phloretin pretreatment reduced the LPS-induced release of IL-6 and IL-8 as well as VEGF. Phloretin increased intracellular levels of reactive oxygen species and nuclear translocation of Nrf2. It also inhibited glucose uptake into ARPE-19 cells and the phosphorylation of Jun-activated kinase (JNK). Subsequent studies revealed that Nrf2, but not the inhibition of glucose uptake or JNK phosphorylation, was the main pathway of phloretin's anti-inflammatory activities. Phloretin was robustly anti-inflammatory in RPE cells and reduced IL-8 secretion via activation of Nrf2 but the evaluation of its potential in the treatment or prevention of AMD requires further studies.

Topics: Epithelial Cells; Glucose; Humans; Inflammation; Interleukin-6; Interleukin-8; Lipopolysaccharides; Macular Degeneration; NF-E2-Related Factor 2; NF-kappa B; Phloretin; Retinal Pigment Epithelium; Retinal Pigments; Vascular Endothelial Growth Factor A

To identify the inflammatory cytokine profile in the aqueous humor (AH) of patients with intraocular inflammation (IOI) after intravitreal administration of brolucizumab (IVBr) for neovascular age-related macular degeneration.. Eight eyes from seven patients with IOI after initial IVBr (IVBrIOI +) were enrolled. Sixteen eyes from 16 patients without IOI after IVBr (IVBrIOI -) and aflibercept (IVA) were used as controls. AH samples were analyzed using a multiplex immunoassay.. C-C motif chemokine ligand (CCL)2, C-X-C motif chemokine ligand (CXCL)1, CXCL10, CXCL13, interleukin (IL)-6, IL-8, IL-10, matrix metalloproteinase (MMP)-1, MMP-9, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), intercellular adhesion molecule (ICAM)-1, E-selectin, and P-selectin levels were significantly higher in IVBrIOI + than in IVBrIOI - and IVA. Vascular endothelial growth factor (VEGF) was significantly lower in IVBrIOI - compared to that in IVBrIOI + and IVA. In the IVBrIOI + group, there were significant correlations between CCL2, CXCL1, IL-6, IL-8, IL-10, G-CSF, GM-CSF, ICAM-1, and E-selectin, which also exhibited significant correlations in the IVBrIOI - group.. The number of inflammatory cytokines increases during IOI, which is associated with type IV hypersensitivity and vascular inflammation. Some cytokines exhibit correlations even in non-inflamed eyes, indicating a subclinical response to IVBr.

Topics: Angiogenesis Inhibitors; Aqueous Humor; Cytokines; E-Selectin; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation; Interleukin-10; Interleukin-6; Interleukin-8; Intravitreal Injections; Ligands; Macular Degeneration; Vascular Endothelial Growth Factor A

Fucoidans from brown algae are described as anti-inflammatory, antioxidative, and antiangiogenic. We tested two

Topics: Animals; Humans; Hydrogen Peroxide; Interleukin-8; Laminaria; Macular Degeneration; Retinal Pigment Epithelium; Swine; Vascular Endothelial Growth Factor A

Age-related macular degeneration (AMD) is a complex eye disease in which decline in autophagy leads to the accumulation of sequestosome 1/p62 (SQSTM1/p62)-labeled waste material inside the retinal pigment epithelial (RPE) cells, and the condition results in activation of the inflammasome signaling and IL-1β secretion. Here, we have studied the role of SQSTM1/p62 in the production of IL-6, IL-8, and MCP-1 in the presence or absence of IL-1β. SQSTM1/p62 was either overexpressed or silenced in ARPE-19 cells, which were then exposed to IL-1β. Alternatively, bafilomycin A was used to demonstrate the functional decline of autophagy with increased SQSTM1/p62 levels. The protein concentration of SQSTM1/p62 was measured using the western blot technique, and interleukin levels were determined by ELISA. In IL-1β-loaded RPE cells, SQSTM1/p62 depletion and overexpression increased the production of MCP-1 and IL-8, respectively. Neither knock-down nor overexpression of SQSTM1/p62 induced the release of IL-6. Our data suggest that SQSTM1/p62 is a significant factor in inflammatory responses, especially following the inflammasome activation.

Topics: Cell Line; Chemokine CCL2; Epithelial Cells; Humans; Inflammasomes; Interleukin-1beta; Interleukin-8; Macrolides; Macular Degeneration; Retinal Pigment Epithelium; Sequestosome-1 Protein

In this study, we elucidated the effects of berberine, a major alkaloid component contained in medicinal herbs, such as Phellodendri Cortex and Coptidis Rhizoma, on expression of monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) in a human retinal pigment epithelial cell line (ARPE-19) caused by lipopolysaccharide (LPS) stimulation.. ARPE-19 cells were cultured to confluence. Berberine and LPS were added to the medium. MCP-1 and IL-8 mRNA were measured by real-time polymerase chain reaction. MCP-1 and IL-8 protein concentrations in the media were measured using enzyme-linked immunosorbent assay.. After stimulation with LPS, MCP-1 and IL-8 mRNA in ARPE-19 cells reached maximum levels at 3 h, and MCP-1 and IL-8 protein in the culture media reached maximum levels at 24 h. Berberine dose-dependently inhibited MCP-1 and IL-8 mRNA expression of the cells and protein levels in the media stimulated with LPS.. These findings indicate that berberine inhibited the expression of MCP-1 and IL-8 induced by LPS.

Topics: Berberine; Cells, Cultured; Chemokine CCL2; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Humans; Interleukin-8; Lipopolysaccharides; Macular Degeneration; Pigment Epithelium of Eye; RNA

Angiopoietin 2 (ANG2) is a proangiogenic cytokine which may have an implication in neovascular age related macular degeneration (nAMD). In 24 eyes of 24 subjects presenting with treatment naïve nAMD and 26 eyes of 26 control patients, aqueous humor samples were collected at the time of intervention (intravitreal injection of anti-vascular endothelial growth factor or cataract extraction). Best corrected visual acuity (BCVA) with and central macular thickness (CMT) using optical coherence tomography (OCT) were measured before each injection in the nAMD group. Aqueous cytokine levels were determined by immunoassay using a multiplex array (Quansys Biosciences, Logan, UT). Levels of ANG2 in the aqueous were significantly higher in nAMD patients than those of the control group (p < 0.0001), so were hepatocyte growth factor (HGF), interleukin-8 (IL-8) and tissue inhibitor of metalloproteinase 1 (TIMP 1), all with p < 0.001. ANG2 correlated with worse BCVA (r = 0.44, p-value = 0.027) and greater CMT (r = 0.66, p-value < 0.0001) on optical coherence tomography (OCT). ANG2 is upregulated in patients with nAMD and correlates with severity of disease at presentation.

Topics: Aged; Aged, 80 and over; Angiopoietin-2; Aqueous Humor; Biomarkers; Case-Control Studies; Female; Hepatocyte Growth Factor; Humans; Interleukin-8; Macular Degeneration; Male; Middle Aged; Tissue Inhibitor of Metalloproteinase-1; Visual Acuity

Phagocytosis of daily shed photoreceptor outer segments is an important function of the retinal pigment epithelium (RPE) and it is essential for retinal homeostasis. RPE dysfunction, especially impairment of its phagocytic ability, plays an essential role in the pathogenesis of age-related macular degeneration (AMD). Statins, or HMG CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors, are drugs with multiple properties that have been extensively used to treat hyperlipidemia. However, their effect on RPE cells has not been fully elucidated. Here we report that high dose atorvastatin increased the phagocytic function of ARPE-19 cells, as well as rescue the cells from the phagocytic dysfunction induced by cholesterol crystals and oxidized low-density lipoproteins (ox-LDL), potentially by increasing the cellular membrane fluidity. Similar effects were observed when evaluating two other hydrophobic statins, lovastatin and simvastatin. Furthermore, atorvastatin was able to block the induction of interleukins IL-6 and IL-8 triggered by pathologic stimuli relevant to AMD, such as cholesterol crystals and ox-LDL. Our study shows that statins, a well-tolerated class of drugs with rare serious adverse effects, help preserve the phagocytic function of the RPE while also exhibiting anti-inflammatory properties. Both characteristics make statins a potential effective medication for the prevention and treatment of AMD.

Topics: Atorvastatin; Cell Line; Cholesterol; Epithelial Cells; Humans; Inflammation; Interleukin-6; Interleukin-8; Lipoproteins, LDL; Macular Degeneration; Phagocytosis; Retinal Pigment Epithelium

Infiltrating immune cells including monocytes/macrophages have been implicated in the pathogenesis of neovascular age-related macular degeneration (nAMD). The aim of this study was to investigate the cytokine and chemokine expression and secretion profile of peripheral blood mononuclear cells (PBMCs) from nAMD patients and the relationship between the cytokine/chemokine expression profile and clinical phenotype of nAMD, including macular fibrosis, macular atrophy or the responsiveness to anti-VEGF therapy.. One hundred sixty-one nAMD patients and 43 controls were enrolled in this study. nAMD patients were divided into subgroups based on the presence/absence of (1) macular atrophy, (2) macular fibrosis and (3) responsiveness to anti-VEGF therapy; 25-30 ml of peripheral blood were obtained from all participants and 5 ml were used for serum collection, and the remaining were used for PBMC isolation using density gradient centrifugation. Intracellular cytokine expressions by PBMCs following phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation were examined using flow cytometry. Cytokine productions in lipopolysaccharides (LPS)-or 1% oxygen -treated PBMC were measured using cytometric bead array (CBA) assay. In addition, cytokine and chemokine levels in the serum were also measured by CBA assay.. PBMCs from nAMD patients secreted higher levels of IL-8, CCL2 and VEGF, especially following LPS and 1% oxygen stimulation, than those from controls. 60~80% of IL-8 producing cells were CD11b. PBMCs, in particular monocytes, may contribute to CNV development in nAMD through secreting elevated levels of IL-8, CCL2 and VEGF after they are recruited to the macula. Apart from VEGF, IL-8 and CCL2 may be additional targets for nAMD management.

Topics: Aged; Aged, 80 and over; Angiogenesis Inhibitors; Cells, Cultured; Chemokine CCL2; Choroidal Neovascularization; Female; Humans; Interleukin-8; Intravitreal Injections; Leukocytes, Mononuclear; Macular Degeneration; Male; Middle Aged; Vascular Endothelial Growth Factor A

To investigate possible associations between VEGFR-2 and IL-8 gene SNPs and 1-year response to intravitreal ranibizumab for exudative age-related macular degeneration.. Sixty-four eyes underwent a loading phase of three monthly intravitreal injections of ranibizumab 0.5 mg/0.05 ml followed by Pro Re Nata retreatment. VEGFR-2 rs2071559 (-604 A/G) and IL-8 rs4073 (-251 A/T) were analyzed.. Ranibizumab was significantly more effective as measured by visual acuity in patients harboring the IL-8 rs4073 TT genotype (p = 0.045), whereas patients carrying the VEGFR-2 rs2071559 CC genotype revealed better functional response as measured by mean retinal sensitivity (p = 0.034).. IL-8 rs4073 and VEGFR-2 rs2071559 genotypes may represent important molecular determinants to modulate final outcomes in neovascular age-related macular degeneration patients.

Topics: Aged; Aged, 80 and over; Angiogenesis Inhibitors; Female; Follow-Up Studies; Fovea Centralis; Genotype; Humans; Interleukin-8; Intravitreal Injections; Macular Degeneration; Male; Middle Aged; Polymorphism, Single Nucleotide; Prospective Studies; Ranibizumab; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Our previous study indicated that Aβ-induced Retinal Pigment Epithelial (RPE) cell senescence may be associated with chronic inflammation in age-related macular degeneration (AMD). The present study was designed to explore whether Aβ deposition and RPE senescence could be found in the senescence-prone mouse strain 8 (SAMP8), which is an animal model for AMD.. Eyes of both SAMP8 and age-matched SAMR1 (SAM resistant) mice were examined in vivo by fundus photography and electroretinography (ERG). Retinal morphological features were assessed using light and electron microscopy. Aβ deposition and p16-positive senescent RPE cells were traced using immunofluorescence labeling. P16 expression was detected using western blot. Expressions of IL-6 and IL-8 in RPE/choroid were analyzed using RT-PCR.. In fundus of SAMP8, age-dependent increase of drusen-like lesions and the increase of granular autofluorescent spots were respectively detected using IR (near-infrared) and AF (autofluorescence) imaging of confocal scanning laser ophthalmoscope. The amplitude of the ERGs declined with age in SAMP8 and these changes were paralleled with the significant changes in retinal morphological features examined by funduscopy. Histopathological analysis found significant loss of photoreceptor outer segments (OS) and abnormal localization of RPE cells in aged SAMP8 mice. Degenerative changes in RPE cells of aged SAMP8 mice, including massive vacuoles, thickened Bruch's membrane (BrM), and loss of basal infoldings were further confirmed by electron microscopy. Increased Aβ deposits in OS layer and p16-positive senescent RPE cells were observed using immunofluorescence microscopy. Western blot confirmed that P16 expression was significantly increased in RPE cells of aged SAMP8 mice. Expressions of proinflammatory IL-6 and IL-8 were significantly upregulated in RPE/choroid of aged SAMP8 mice.. Our results showed that aged SAMP8 mice developed ocular pathology similar to some features of human AMD. In this AMD mouse model, Aβ deposition and RPE senescence may be associated with AMD development, and RPE senescence is likely a mechanistic link between Aβ deposition and inflammation.

Topics: Age Factors; Amyloid beta-Peptides; Animals; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p16; Disease Models, Animal; Electroretinography; Epithelial Cells; Fundus Oculi; Humans; Immunohistochemistry; Interleukin-6; Interleukin-8; Macular Degeneration; Mice; Microscopy, Electron, Transmission; Photography; Retinal Pigment Epithelium

Retinal pigment epithelial (RPE) cells can undergo different forms of cell death, including autophagy-associated cell death during age-related macular degeneration (AMD). Failure of macrophages or dendritic cells (DCs) to engulf the different dying cells in the retina may result in the accumulation of debris and progression of AMD. ARPE-19 and primary human RPE cells undergo autophagy-associated cell death upon serum depletion and oxidative stress induced by hydrogen peroxide (H2O2). Autophagy was revealed by elevated light-chain-3 II (LC3-II) expression and electron microscopy, while autophagic flux was confirmed by blocking the autophago-lysosomal fusion using chloroquine (CQ) in these cells. The autophagy-associated dying RPE cells were engulfed by human macrophages, DCs and living RPE cells in an increasing and time-dependent manner. Inhibition of autophagy by 3-methyladenine (3-MA) decreased the engulfment of the autophagy-associated dying cells by macrophages, whereas sorting out the GFP-LC3-positive/autophagic cell population or treatment by the glucocorticoid triamcinolone (TC) enhanced it. Increased amounts of IL-6 and IL-8 were released when autophagy-associated dying RPEs were engulfed by macrophages. Our data suggest that cells undergoing autophagy-associated cell death engage in clearance mechanisms guided by professional and non-professional phagocytes, which is accompanied by inflammation as part of an in vitro modeling of AMD pathogenesis.

Topics: Adenine; Autophagy; Biomarkers; Cell Line; Chloroquine; Coculture Techniques; Culture Media, Serum-Free; Gene Expression; Genes, Reporter; Green Fluorescent Proteins; Humans; Hydrogen Peroxide; Interleukin-6; Interleukin-8; Macrophages; Macular Degeneration; Microtubule-Associated Proteins; Models, Biological; Oxidative Stress; Phagocytosis; Primary Cell Culture; Retinal Pigment Epithelium; Triamcinolone

Calcium signaling is an important intracellular pathway. Increased intracellular calcium is associated with cytokine regulation and inflammatory signals secretion. The purpose of this study is to understand the molecular mechanisms by which calcium signaling controls IL-8 activation in human RPE cells.. Fluorescence-based calcium imaging and different mutants of IL-8 plasmids were used in this study. The IL-8 promoter activation, gene expression, and secretion were detected by using luciferase reporter assay, quantitative real-time PCR (Q-PCR), and ELISA, respectively. In addition, pharmacological inhibitors and small interfering RNA (siRNA) were applied to clarify the mechanisms of IL-8 activation.. Our study reported that intracellular calcium mobilization activated IL-8 gene expression and secretion. Application of pharmacological inhibitor BAY 11-7082, siRNA, and plasmids of the nuclear factor κ light chain enhancer of activated B cells (NF-κB) binding site, we identified that NF-κB is the main transcription factor involved in intracellular calcium mobilization-mediated IL-8 activation in human RPE cells.. Collectively, our findings highlight the important role of intracellular calcium mobilization in the activation of IL-8. These findings may be helpful for the clinical applications in the age-related macular degeneration (AMD) prevention and treatment.

Topics: Blotting, Western; Calcium; Calcium Signaling; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Humans; Interleukin-8; Macular Degeneration; Real-Time Polymerase Chain Reaction; Retinal Pigment Epithelium; RNA

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly of industrialized nations, and there is increasing evidence to support a role for chronic inflammation in its pathogenesis. Mitochondrial DNA (mtDNA) has been recently reported to be pro-inflammatory in various diseases such as Alzheimer's and heart failure. Here, we report that intracellular mtDNA induces ARPE-19 cells to secrete inflammatory cytokines IL-6 and IL-8, which have been consistently associated with AMD onset and progression. The induction was dependent on the size of mtDNA, but not on specific sequence. Oxidative stress plays a major role in the development of AMD, and our findings indicate that mtDNA induces IL-6 and IL-8 more potently when oxidized. Cytokine induction was mediated by STING (Stimulator of Interferon Genes) and NF-κB as evidenced by abrogation of the cytokine response with the use of specific inhibitors (siRNA and BAY 11-7082, respectively). Finally, mtDNA primed the NLRP3 inflammasome. This study contributes to our understanding of the potential pro-inflammatory role of mtDNA in the pathogenesis of AMD.

Topics: Carrier Proteins; Cell Line; DNA, Mitochondrial; Humans; Inflammasomes; Interleukin-6; Interleukin-8; Macular Degeneration; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress

To study the association of serum levels of inflammatory mediators and angiogenic factors with genetic polymorphism in Pakistani age-related macular degeneration (AMD) patients.. This was a cross-sectional and case-control study that included 90 AMD patients diagnosed through slit-lamp examination, fundoscopy, and ocular coherence tomography. For reference and comparison purposes, 100 healthy age-matched subjects (controls) were also recruited. IL-6, IL-8, VEGF, and CRP levels were estimated in the serum samples of patients and control subjects. Using restriction fragment length polymorphism, single nucleotide polymorphisms were studied in IL-6 (rs1800795, rs1800796, rs1800797), IL-8 (rs4073, rs2227306, rs2227543), VEGF (rs3025039, rs699947), and CRP genes (rs1205, rs1130864). Since the data were obtained from a sample population, the Box-Cox transformation algorithm was applied to reduce heterogeneity of error. Multivariate analyses of variance (M-ANOVA) were applied on the transformed data to investigate the association of serum levels of IL-6, IL-8, VEGF, and CRP with AMD. Genotype and allele frequencies were compared through χ(2) tests applying Hardy-Weinberg equilibrium. The serum concentrations of IL-6 and IL-8, VEGF, and CRP between homozygotes and heterozygotes were compared through one-way ANOVA. Significance level was p<0.05.. Compared to control subjects, serum IL-6 (p<0.0001), IL-8 (p<0.0001), VEGF (p<0.0001), and CRP (p<0.0001) levels were significantly elevated in the AMD patients. For rs1800795, patients with the GG genotype showed significantly raised levels of IL-6 compared to those with GC and CC genotypes (p<0.0001). Serum IL-8 levels were significantly higher in patients with the GG genotype compared to the GC and CC genotypes for the single nucleotide polymorphism (SNP) rs2227543 (p<0.002). Similarly, significantly higher VEGF levels were detected for genotype TT for rs3025039 SNP (p<0.038). However, no significant alteration in serum CRP levels was detected in hetero- or homozygotes for rs1205 and rs1130864 SNPs.. Serum IL-6, IL-8, and VEGF levels are substantially increased in AMD, and the levels coincide with polymorphism in the respective gene. No such relationship appears to exist with regard to SNPs of CRP.

Topics: Aged; Aged, 80 and over; Angiogenic Proteins; C-Reactive Protein; Case-Control Studies; Cross-Sectional Studies; Female; Genetic Association Studies; Heterozygote; Homozygote; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Macular Degeneration; Male; Middle Aged; Pakistan; Polymorphism, Single Nucleotide; Vascular Endothelial Growth Factor A

The ubiquitin-proteasome pathway (UPP) plays an important role in regulating gene expression. Retinal pigment epithelial cells (RPE) are a major source of ocular inflammatory cytokines. In this work we determined the relationship between impairment of the UPP and expression of inflammation-related factors. The UPP could be impaired by oxidative stress or chemical inhibition. Impairment of the UPP in RPE increased the expression of several inflammatory cytokines, such as IL-6 and IL-8. However, the expression of monocyte chemoattractant protein-1 (MCP-1) and complement factor H (CFH) and was reduced upon impairment of the UPP. These data suggest that impairment of the UPP in RPE may be one of the causes of retinal inflammation and abnormal functions of monocyte and the complement system during the pathogenesis of age-related macular degeneration.

Topics: Cell Line; Chemokine CCL2; Complement Factor H; Humans; Interleukin-6; Interleukin-8; Macular Degeneration; Oxidative Stress; Proteasome Endopeptidase Complex; Retinal Pigment Epithelium; Retinitis; Ubiquitin

Chronic inflammation is implicated in the pathogenesis of AMD. The source of chronic inflammation is often attributed to the progressive activation of immune cells over time. However, recent studies have shown that senescent cells can alter tissue microenvironment via secretion of growth factors, proteases, and inflammatory cytokines and might be an additional source of chronic inflammation. We hypothesized that altered secretory pattern in Aβ-induced senescent RPE cells may contribute to compromised RPE barrier integrity and chronic inflammation in AMD.. Senescence was assessed by measuring the SA-β-Galactosidase activity, the expressions of p16(INK4a) and ATM, and cell cycle analysis. Expressions of IL-8 and MMPs were analyzed by RT-PCR, ELISA, and gelatin zymography. The barrier structures of RPE cells were detected by actin-tracker, ZO-1, claudin-19, occludin immunochemistry, and Western blot; barrier function was analyzed by measuring transepithelial resistance (TER) and transepithelial diffusion rate of FITC-dextran. For inhibitory studies, MMP-9 was inhibited by RNA interference strategy.. Aβ promotes RPE cells to enter senescence and secrete higher concentrations of IL-8 and MMP-9. Secretion of MMP-9 is associated with compromised barrier integrity and with processing of IL-8 to a more activated form. Silence of MMP-9 preserved the barrier integrity of senescent RPE cells.. The altered secretory phenotype of senescent RPE cells may contribute to age-related inflammation in AMD. Chinese Abstract.

Topics: Amyloid beta-Peptides; Cellular Microenvironment; Cellular Senescence; Chronic Disease; Epithelial Cells; Fetus; Humans; Interleukin-8; Macular Degeneration; Matrix Metalloproteinase 9; Occludin; Peptide Fragments; Retinal Pigment Epithelium; Retinitis; RNA, Small Interfering; Zonula Occludens-1 Protein

Age-related macular degeneration (AMD) is the main cause of blindness in the developed world. The etiology of AMD is multifactorial due to the interaction between genetic and environmental factors. IL-8 has a role in inflammation and angiogenesis; we report the genetic characterization of IL-8 allele architecture and evaluate the role of SNPs or haplotypes in the susceptibility to wet AMD, case-control study.. Case-control study including 721 AMD patients and 660 controls becoming from Italian population. Genotyping was carried out by Real Time-PCR. Differences in the frequencies were estimated by the chi-square test. Direct sequencing was carried out by capillary electrophoresis trough ABI3130xl.. rs2227306 showed a p-value of 4.15*10(-5) and an Odds Ratio (OR) for T allele of 1.39 [1.19-1.62]. After these positive results, we sequenced the entire IL-8 regulatory and coding regions of 60 patients and 30 controls stratified for their genotype at rs2227306. We defined two different haplotypes involving rs4073 (A/T), rs2227306 (C/T), rs2227346 (C/T) and rs1126647 (A/T): A-T-T-T (p-value: 2.08*10(-9); OR: 1.68 [1.43-1.97]) and T-C-C-A (p-value: 7.07*10(-11); OR: 0.60 [0.51-0.70]). To further investigate a potential functional role of associated haplotypes, we performed an expression study on RNA extracted from whole blood of 75 donors to verify a possible direct correlation between haplotype and gene expression, failing to reveal significant differences.. These results suggest a possible secondary role of IL-8 gene in the development of the disease. This paper outlines the importance of association between inflammation and AMD. Moreover IL-8 is a new susceptibility genomic biomarker of AMD.

Topics: Aged; Aged, 80 and over; Case-Control Studies; Female; Gene Expression Regulation; Genetic Association Studies; Genetic Predisposition to Disease; Haplotypes; Humans; Interleukin-8; Macular Degeneration; Male; Middle Aged; Odds Ratio; Polymorphism, Single Nucleotide

Disturbances in cholesterol metabolism and increased levels of cholesterol oxidation products (oxysterols) in retina may contribute to age-related macular degeneration (AMD). The role of oxysterols or of their target receptors liver X receptors (LXRs) and estrogen receptors (ERs) in the pathogenesis of MD is ill-known. The purpose of this study is to determine the extent to which the oxysterols 27-hydroxycholesterol (27-OHC), 25-hydroxycholesterol (25-OHC) and 7-ketocholesterol (7-KC) affect the transcriptional activity of LXR and ER.. ARPE-19 cells, untreated or incubated with 27-OHC, 25-OHC or 7-KC for 24 h were harvested. We used Western blot analyses for detecting ERs and LXRs expression, dual luciferase assays for measuring LXRs and ERs transcriptional activity, cytotox-ONE homogeneous membrane integrity assay for measuring cytotoxicity, JC-1 method for measuring mitochondrial membrane potential changes and ELISA for measuring cytokine levels.. Both LXRs and ERs are expressed and are transcriptionally active in ARPE-19 cells. 27-OHC, 25-OHC and 7-KC inhibited ER-mediated transcriptional activity, whereas 27-OHC and 25-OHC increased LXR-mediated transcription. E2 reduced 25-OHC and 27-OHC-induced cytotoxicity, mitochondrial permeability potential decline, and cytokine secretion. The LXR agonist GW3965 or the LXR antagonist 5α-6α-epoxycholesterol-3-sulfate (ECHS) did not offer protection against either 27-OHC and 25-OHC or 7-KC.. Increased levels of oxysterols can decrease ER and increase LXR signaling. ER agonists can offer protection against cytotoxic effects of 27-OHC and 25-OHC, two oxysterols derived by enzymatic reactions. Although they exert similar toxicity, the cellular mechanisms involved in the toxic effects of oxysterols whether derived by enzymatic or autoxidation reactions appear to be different.

Topics: Cell Line; Chemokine CCL2; Drug Interactions; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Humans; Hydroxycholesterols; Interleukin-6; Interleukin-8; Ketocholesterols; Liver X Receptors; Macular Degeneration; Orphan Nuclear Receptors; Oxidation-Reduction; Platelet-Derived Growth Factor; Retinal Pigment Epithelium; Transcription, Genetic; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

To investigate Toll-like receptor (TLR) expression and reactivity in patients with the wet form age-related macular degeneration (AMD).. Blood samples were collected from 25 patients with wet AMD and 25 age-matched healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-Hypaque density gradient centrifugation. Expression of TLR1 to TLR10 mRNAs in PBMCs from 15 patients with wet AMD and 15 controls was assessed with real-time PCR. TLR2 and TLR3 protein levels in PBMCs from six patients with wet AMD and six controls were measured with flow cytometry. After PBMCs were stimulated with peptidoglycan (PGN) and poly(I:C), the specific ligands of TLR2 and TLR3, cytokines interleukin-6 (IL-6), IL-8, VEGF, and monocyte chemoattractant protein-1 (MCP-1) production in 11 patients with wet AMD and 11 controls were assessed.. TLR2 and TLR3 mRNA and protein expression in the PBMCs of the patients with wet AMD was significantly higher than that in the controls. However, the difference in TLR1 and TLR4-10 mRNA expression between the two groups was not significant. The PBMCs of the patients with wet AMD produced more IL-6 and IL-8 proteins than the controls in response to PGN, a ligand for TLR2, and more IL-6 protein than the controls in response to poly(I:C), the ligand for TLR3. However, there was no significant difference in vascular endothelial growth factor and monocyte chemoattractant protein-1 production between the wet AMD group and the control group when the PBMCs were stimulated with PGN or poly(I:C).. Our data suggested that upregulation of TLR2 and TLR3 may be associated with the pathogenesis of wet AMD.

Topics: Cell Membrane; Cohort Studies; Humans; Interleukin-6; Interleukin-8; Leukocytes, Mononuclear; Ligands; Macular Degeneration; RNA, Messenger; Toll-Like Receptor 2; Toll-Like Receptor 3

Chronic inflammation induced by amyloid-beta (Aβ) plays a key role in the development of age-related macular degeneration (AMD), and matrix metalloproteinase-9 (MMP-9), interleukin (IL)-6, and IL-8 may be associated with chronic inflammation in AMD. Sirtuin 1 (SIRT1) regulates inflammation via inhibition of nuclear factor-kappa B (NF-κB) signaling, and resveratrol has been reported to prevent Aβ-induced retinal degeneration; therefore, we investigated whether this action was mediated via activation of SIRT1 signaling. Human adult retinal pigment epithelial (RPE) cells were exposed to Aβ, and overactivation and knockdown of SIRT1 were performed to investigate whether SIRT1 is required for abrogating Aβ-induced inflammation. We found that Aβ-induced RPE barrier disruption and expression of IL-6, IL-8, and MMP-9 were abrogated by the SIRT1 activator SRT1720, whereas alterations induced by Aβ in SIRT1-silenced RPE cells were not attenuated by SRT1720. In addition, SRT1720 inhibited Aβ-mediated NF-κB activation and decrease of the NF-κB inhibitor, IκBα. Our findings suggest a protective role for SIRT1 signaling in Aβ-dependent retinal degeneration and inflammation in AMD.

Topics: Adult; Amyloid beta-Peptides; Antioxidants; Blood-Retinal Barrier; Cell Survival; Enzyme Assays; Gene Silencing; Heterocyclic Compounds, 4 or More Rings; Humans; Inflammation; Interleukin-6; Interleukin-8; Macular Degeneration; Matrix Metalloproteinase 9; NF-kappa B; Primary Cell Culture; Real-Time Polymerase Chain Reaction; Resveratrol; Retinal Pigment Epithelium; RNA Interference; Signal Transduction; Sirtuin 1; Stilbenes

Age-related macular degeneration (AMD) is the predominant cause of irreversible blindness in the elderly population. Despite intensive basic and clinical research, its pathogenesis remains unclear. However, evidence suggests that immunological and inflammatory factors contribute to the pathogenesis of AMD. A newly identified cytokine, IL-33, appears to be an important pro-inflammatory cytokine promoting tissue inflammation. In this study, IL-33 was increased through amyloid-beta(1-40) (Aβ(1-40)) stimulation and regulated inflammatory cytokines including IL-6, IL-8, IL-1β, and TNF-α secretion using different signaling pathways in retinal pigment epithelium (RPE) cells. Furthermore, ST2L, the important component of the IL-33 receptor, was significantly increased following recombinant human IL-33 stimulation in RPE cells. These findings suggest that IL-33-mediated inflammatory responses in RPE cells are involved in the pathogenesis of AMD. Greater understanding of the inflammatory effect of IL-33 and its role in RPE cells should aid the development of future clinical therapeutics and enable novel pharmacological approaches towards the prevention of AMD.

Topics: Amyloid beta-Peptides; Anthracenes; Butadienes; Cell Line; Humans; Imidazoles; Inflammation; Interleukin-1 Receptor-Like 1 Protein; Interleukin-1beta; Interleukin-33; Interleukin-6; Interleukin-8; Interleukins; JNK Mitogen-Activated Protein Kinases; Macular Degeneration; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Nitriles; Peptide Fragments; Pyridines; Receptors, Cell Surface; Retinal Pigment Epithelium; Sulfones; Tumor Necrosis Factor-alpha

Topics: Animals; Cells, Cultured; Dietary Supplements; Interleukin-6; Interleukin-8; Lipopolysaccharides; Lutein; Macrophages; Macular Degeneration; Mice; Mice, Inbred C57BL; Oxidative Stress; Retinal Pigment Epithelium; Tumor Necrosis Factor-alpha; Xanthophylls; Zeaxanthins

Oxidative damage and inflammation are related to the pathogenesis of age-related macular degeneration (AMD). Epidemiologic studies suggest that insufficient dietary lutein and zeaxanthin intake or lower serum zeaxanthin levels are associated with increased risk for AMD. The objective of this work is to test the protective effects of lutein and zeaxanthin against photooxidative damage to retinal pigment epithelial cells (RPE) and oxidation-induced changes in expression of inflammation-related genes. To mimic lipofuscin-mediated photooxidation in vivo, we used ARPE-19 cells that accumulated A2E, a lipofuscin fluorophore and photosensitizer, as a model system to investigate the effects of lutein and zeaxanthin supplementation. The data show that supplementation with lutein or zeaxanthin in the medium resulted in accumulation of lutein or zeaxanthin in the RPE cells. The concentrations of lutein and zeaxanthin in the cells were 2- to 14-fold of that detected in the medium, indicating that ARPE-19 cells actively take up lutein or zeaxanthin. As compared with untreated cells, exposure of A2E-containing RPE to blue light resulted in a 40-60% decrease in proteasome activity, a 50-80% decrease in expression of CFH and MCP-1, and an~20-fold increase in expression of IL-8. The photooxidation-induced changes in expression of MCP-1, IL-8, and CFH were similar to those caused by chemical inhibition of the proteasome, suggesting that inactivation of the proteasome is involved in the photooxidation-induced alteration in expression of these inflammation-related genes. Incubation of the A2E-containing RPE with lutein or zeaxanthin prior to blue light exposure significantly attenuated the photooxidation-induced inactivation of the proteasome and photooxidation-induced changes in expression of MCP-1, IL-8, and CFH. Together, these data indicate that lutein or zeaxanthin modulates inflammatory responses in cultured RPE in response to photooxidation. Protecting the proteasome from oxidative inactivation appears to be one of the mechanisms by which lutein and zeaxanthin modulate the inflammatory response. Similar mechanisms may explain salutary effects of lutein and zeaxanthin in reducing the risk for AMD.

Topics: Cells, Cultured; Chemokine CCL2; Complement Factor H; Culture Media; Dietary Supplements; Down-Regulation; Gene Expression; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Lutein; Macular Degeneration; Oxidation-Reduction; Oxidative Stress; Photochemical Processes; Proteasome Endopeptidase Complex; Radiation-Protective Agents; Retinal Pigment Epithelium; Ultraviolet Rays; Xanthophylls; Zeaxanthins

There is evidence for complement dysfunction in age-related macular degeneration (AMD). Complement activation leads to formation of the membrane attack complex (MAC), known to assemble on retinal pigment epithelial (RPE) cells. Therefore, the effect of sub-lytic MAC on RPE cells was examined with regard to pro-inflammatory or pro-angiogenic mediators relevant in AMD.. For sub-lytic MAC induction, RPE cells were incubated with an antiserum to complement regulatory protein CD59, followed by normal human serum (NHS) to induce 5% cell death, measured by a viability assay. MAC formation was evaluated by immunofluorescence and FACS analysis. Interleukin (IL)-6, -8, monocytic chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF) were quantified by enzyme-linked immunosorbent assay (ELISA). Intracellular MCP-1 was analysed by immunofluorescence, vitronectin by western blotting, and gelatinolytic matrix metalloproteinases (MMPs) by zymography.. Incubation of RPE cells with the CD59 antiserum followed by 5% NHS induced sub-lytic amounts of MAC, verified by FACS and immunofluorescence. This treatment stimulated the cells to release IL-6, -8, MCP-1, and VEGF. MCP-1 staining, production of vitronectin, and gelatinolytic MMPs were also elevated in response to sub-lytic MAC.. MAC assembly on RPE cells increases the IL-6, -8, and MCP-1 production. Therefore, sub-lytic MAC might have a significant role in generating a pro-inflammatory microenvironment, contributing to the development of AMD. Enhanced vitronectin might be a protective mechanism against MAC deposition. In addition, the increased expression of gelatinolytic MMPs and pro-angiogenic VEGF may be associated with neovascular processes and late AMD.

Topics: Cell Line; Chemokine CCL2; Complement Activation; Complement Membrane Attack Complex; Humans; Immunohistochemistry; Interleukin-6; Interleukin-8; Macular Degeneration; Matrix Metalloproteinases; Retinal Pigment Epithelium; Vascular Endothelial Growth Factor A; Vitronectin

: To evaluate the concentration of various cytokines in the aqueous humor of patients with naive, recurrent, and regressed choroidal neovascularization (CNV) of age-related macular degeneration after bevacizumab treatment.. : Aqueous humor samples were collected from 36 eyes with age-related macular degeneration and 10 controls during cataract surgery. Of 36 patients with age-related macular degeneration, 5 eyes were naïve to bevacizumab injection, 14 eyes had recurrent CNV after bevacizumab treatment, and 17 eyes had regressed CNV after bevacizumab treatment. Cytokines were measured by an immunoassay using multianalyte biochip array technology (Evidence investigator cytokine and growth factor biochip array, RANDOX laboratories Ltd., Crumlin, UK).. : No significant difference in the cytokine levels was noted between the control group and the naïve CNV group (all P > 0.05). Vascular endothelial growth factor in both naive (66.8 +/- 35.1 pg/mL) and recurrent CNV groups (55.7 +/- 63.0 pg/mL) was significantly higher compared with regressed CNV group (9.8 +/- 12.8 pg/mL, P = 0.025 and P = 0.004, respectively) but was not statistically different from the control group (81.8 +/- 43.7 pg/mL, P = 0.310 and P = 0.212, respectively). The aqueous humor level of tumor necrosis factor-alpha and interleukin (IL)-2 was significantly lower in recurrent CNV group (P = 0.036 and P = 0.019) compared with the control group. In the active CNV patients (recurrent and naïve CNV groups), the aqueous humor levels of IL-6 and IL-8 significantly correlated with the size of CNV (rho = 0.692, P = 0.001 and rho = 0.745, P < 0.001, respectively).. : Levels of Vascular endothelial growth factor measured in the aqueous humor were significantly related to the disease activity of CNV in age-related macular degeneration. Moreover, IL-2, IL-6, IL-8, and tumor necrosis factor-alpha may be related to the activity of CNV.

Topics: Aged; Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Aqueous Humor; Bevacizumab; Choroidal Neovascularization; Cytokines; Female; Humans; Interleukin-2; Interleukin-6; Interleukin-8; Macular Degeneration; Male; Middle Aged; Osmolar Concentration; Recurrence; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, targets the retinal pigment epithelium (RPE), a monolayer of cells at the back of the eye. As AMD progresses, it can develop into two distinct forms of late AMD: "dry," atrophic AMD, characterized by RPE senescence and geographic RPE loss, and "wet," neovascular AMD, characterized by RPE activation with abnormal growth of choroidal vessels. The genetic and molecular pathways that lead to these diverse phenotypes are currently under investigation. We have found that bone morphogenetic protein-4 (BMP4) is differentially expressed in atrophic and neovascular AMD. In atrophic AMD, BMP4 is highly expressed in RPE, and mediates oxidative stress induced RPE senescencein vitro via Smad and p38 pathways. In contrast, in neovascular AMD lesions, BMP4 expression in RPE is low, possibly a result of local expression of pro-inflammatory mediators. Thus, BMP4 may be involved in the molecular switch determining which phenotypic pathway is taken in the progression of AMD.

Topics: Aging; Bone Morphogenetic Protein 4; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p21; Humans; Hydrogen Peroxide; Interleukin-8; Macular Degeneration; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Retinal Neovascularization; Retinal Pigment Epithelium; Smad Proteins

To determine whether four expression-related cytokine polymorphisms are associated with age-related macular degeneration (AMD).. DNA from 478 cases with AMD and 555 normal controls was genotyped for the pro-inflammatory IL1beta -511C/T, IL6 -174C/G, IL8 -251A/T and anti-inflammatory IL10 -1082G/A cytokine polymorphisms using the 5' nuclease TaqMan(R) assay for allelic discrimination. Associations with AMD were analysed using allelic frequencies.. The -251A allele of the IL8 promoter gene polymorphism was more prevalent in AMD patients than controls (p = 0.037, OR = 1.21, 95% CI = 1.01 to 1.44). Adjusting for age, sex, body mass index (BMI), current smoking and past smoking status did not alter the AMD association significantly (corrected p value = 0.043, OR = 1.23, 95% CI = 1.0 to 1.50).. The pro-inflammatory homozygous IL8 -251AA genotype is an important risk factor for AMD. This may have implications for future therapy with biological agents that could target this cytokine.

Topics: Aged; Aged, 80 and over; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Interleukin-8; Macular Degeneration; Male; Middle Aged; Polymorphism, Single Nucleotide; Risk Factors

Topics: Aged; Genetic Predisposition to Disease; Humans; Inflammation; Interleukin-8; Macular Degeneration; Polymorphism, Genetic

Age-related macular degeneration (AMD) is a leading cause of blindness in the United States, and increasing evidence suggests that it is an inflammatory disease. The prokaryotic obligate intracellular pathogen Chlamydia pneumoniae is emerging as a novel risk factor in cardiovascular disease, and recent sero-epidemiological data suggest that C. pneumoniae infection is also associated with AMD. In this study, we examined choroidal neovascular membrane (CNV) tissue from patients with neovascular AMD for the presence of C. pneumoniae and determined whether the pathogen can dysregulate the function of key cell types in ways that can cause neovascular AMD. Nine CNV removed from patients with neovascular AMD were examined for the presence of C. pneumoniae by immunohistochemistry (IHC) and polymerase chain reaction (PCR); in addition, we performed PCR on nine non-AMD eyes, and IHC on five non-AMD CNV, seven non-AMD eyes, and one internal limiting membrane specimen. Finally, human monocyte-derived macrophages and retinal pigment epithelial (RPE) cells were exposed to C. pneumoniae and assayed in vitro for the production of pro-angiogenic immunomodulators (VEGF, IL-8, and MCP-1). C. pneumoniae was detected in four of nine AMD CNV by IHC and two of nine AMD CNV by PCR, induced VEGF production by human macrophages, and increased production of IL-8 and MCP-1 by RPE cells. In contrast, none of the 22 non-AMD specimens showed evidence for C. pneumoniae. These data indicate that a pathogen capable of inducing chronic inflammation and pro-angiogenic cytokines can be detected in some AMD CNV, and suggest that infection may contribute to the pathogenesis of AMD.

Topics: Adult; Aged; Aged, 80 and over; Cells, Cultured; Chemokine CCL2; Child; Chlamydophila Infections; Chlamydophila pneumoniae; Choroidal Neovascularization; DNA, Bacterial; Eye Infections, Bacterial; Female; Humans; Immunoenzyme Techniques; Interleukin-8; Macrophages; Macular Degeneration; Male; Middle Aged; Pigment Epithelium of Eye; Polymerase Chain Reaction; Vascular Endothelial Growth Factor A