Peripheral blood mononuclear cells from neovascular age-related macular degeneration patients produce higher levels of chemokines CCL2 (MCP-1) and CXCL8 (IL-8)

Abstract

Background: 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.

Methods: 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.

Results: 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⁺CD3^- monocytes. The percentage of CD11b⁺CD3^- IL-8⁺ was significantly increased in nAMD patients compared to controls. PBMCs from nAMD patients without macular fibrosis produced the highest levels of IL-8 and CCL2, whilst PBMCs from nAMD patients with macular atrophy produced highest levels of VEGF. In addition, PBMCs from patients who partially responded to anti-VEGF produced higher levels of IL-8 compared to the cells from complete responders. Interestingly, serum level of CCL2 was not increased in nAMD patients although there was a trend of increased IL-8 in nAMD patients.

Conclusions: 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.

Keywords: Age-related macular degeneration; CCL2; Chemokines; Choroidal neovascularisation; Cytokines; Fibrosis; IL-8.

Fig. 1

Cytokine secretion by PBMC. IL-10, TNFα, CCL2, IL-6, IL-8 and VEGF secretion in the supernatant of PBMCs from controls and nAMD patients under non-stimulated conditions (a), following LPS treatment (b) and under hypoxic conditions (c). Controls n = 28, nAMD n = 75; mean + SEM; #P < 0.05, ##P < 0.01 in univariate analysis using independent samples t test; *P < 0.05, **P < 0.01 in multivariate analysis using multinomial logistic regression

Fig. 2

IL-8 producing PBMCs under non-stimulated conditions and after stimulation with PMA/ionomycin. Percentage of total IL-8 producing PBMCs from controls and nAMD patients (a) and composition of IL-8 producing cells from all samples (b). Percentage of CD11b⁺CD3^- cells that were positive for intracellular IL-8 from controls and nAMD patients (c). Controls n = 27, nAMD = 28; mean + SEM; #P < 0.05, ##P < 0.01 in univariate analysis suing independent samples t test; *P < 0.05 in multivariate analysis using multinomial logistic regression

Fig. 3

IL-6 producing PBMCs under non-stimulated conditions and after stimulation with PMA/ionomycin. Percentage of total (a) and CD11b⁺CD3^- (b) IL-6 producing PBMCs from controls and nAMD patients. Controls n = 27, nAMD = 28; mean + SEM; ##P < 0.01 in univariate analysis using independent samples t test; *P < 0.05 in multivariate analysis using multinomial logistic regression

Fig. 4

Cytokine production and macular fibrosis. CCL2, IL-8 and VEGF levels in PBMC supernatants under non-stimulated condition (a), following LPS treatment (b) and under hypoxic condition (c) as well as percentage of total IL-8 producing PBMCs under non-stimulated conditions (d) from healthy controls and nAMD patients without and with fibrosis. Supernatants: controls, n = 28, fibrosis (−), n = 54, fibrosis (+), n = 21; PBMC: controls, n = 27, fibrosis (−), n = 15, fibrosis (+), n = 13; mean + SEM; #P < 0.05, ##P < 0.01, ###P < 0.001 in univariate analysis using one-way ANOVA; *P < 0.05, **P < 0.01 in multivariate analysis using multinomial logistic regression

Fig. 5

Cytokine production and macular atrophy. CCL2, IL-8 and VEGF levels in PBMC supernatants from healthy controls and nAMD patients without and with macular atrophy (MA) under non-stimulated condition (a), following LPS treatment (b) and under hypoxic condition (c). Controls n = 28, MA (−) n = 17, MA (+) n = 35; mean + SEM; #P < 0.05, ##P < 0.01 in univariate analysis using one-way ANOVA. *P < 0.05, **P < 0.01 in multivariate analysis using multinomial logistic regression

Fig. 6

IL-8 producing PBMC and response to anti-VEGF therapy. Percentage of IL-8 producing PBMCs of controls and nAMD patients not, partially and completely responding to anti-VEGF therapy  under non-stimulated condition (a), and following PMA/ionomycin stimulation (b). Controls:n = 27, non-responders: n = 2, partial responders: n = 16, complete responders: n = 10; mean + SEM; #P < 0.05, in univariate analysis using independent samples t test