CX3CL1/CX3CR1 and CCL2/CCR2 chemokine/chemokine receptor complex in patients with AMD

Abstract

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.

Figure 1. Gating strategy and quantification of CD8⁺ T cells and their expression of CX3CR1.

A, Lymphocytes were identified based on forward scatter and side scatter. B, the lymphocytes were further gated based on their CD4 expression. C, The subset of CD4^- T cells that are CD8 negative is found. D, The fraction of CX3CR1⁺ cells within the CD4⁻ CD8⁺ T cells was calculated. Histogram showing the percentage of CD8⁺ T cells positive for CX3CR1 (dark grey) and the negative isotype control set at 1% (light grey).

Figure 2. Expression of CX3CR1 and CCR2 on CD8+ cells.

A: Percentage of CD8⁺ cells expressing CX3CR1 in the 150 patients included in the study. The dots represent medians and the lines denote inter quartile ranges (IQR). P-values, Mann-Whitney U Test. B: Percentage of CD8⁺ cells expressing CCR2 in the 150 patients included in the study. The dots represent medians and the lines denote inter quartile ranges (IQR).

Figure 3. Correlation between expression of CCR2 and CX3CR1 on CD8⁺ cells.

The plot shows a positive correlation between CCR2+ and CX3CR1 expression on CD8⁺ cells. The majority of participants with combined low frequency of CCR2 and CX3CR1 on CD8 cells were diagnosed with AMD. Combined CCR2 and CX3CR1 receptor expression data was available in 102 patients.

Figure 4. CCR2 expression and clinical response to anti-VEGF treatment.

The patients with neovascular AMD were divided into groups according to change in visual acuity after the initial three anti-VEGF injections. The “Gain of more than 10 ETDRS letters” group consisted of 17 patients, the “VA change (gain or loss) of maximum 10 ETDRS letters” group consisted of 57 patient while the “loss of more than 10 ETDRS letters” group consisted of 12 patients. Percentage of CD4⁺ cells expressing CX3CR1 were calculated for each group. There was a tendency towards a higher expression of CCR2 on CD4+ cells in the group with a VA loss>10 ETDRS letters even though no significant change was found between the groups (p = 0.056, Kruskal-Wallis). The dots represent medians and the lines denote inter quartile ranges (IQR).