Effect of intravitreal ranibizumab injection on aqueous humour cytokine levels in patients with diabetic macular oedema.

Abstract

There is evidence to indicate that inflammatory cytokines, as well as vascular endothelial growth factor (VEGF), play an important role in the development of diabetic macular oedema (DME). Recently, intravitreally injected ranibizumab (IVR) (Lucentis; Novartis, Basel, Switzerland and Genentech Inc., South San Francisco, CA, USA) has been shown to suppress inflammatory cytokines and chemokines that promote adhesion to vascular endothelium or mobilize inflammatory cells by inhibiting multiple isoforms of VEGF-A (Ferrara et al. 2006). Campochiaro et al. (2009) reported that VEGF levels in the aqueous humour were significantly higher in patients with DME (528 ± 112 pg/ml) than in patients with branch retinal vein occlusion (35 ± 3 pg/ml). Muether et al. (2014) measured VEGF levels in the aqueous humour of DME patients receiving IVR, using Luminex, and reported that VEGF levels were completely suppressed in all patients receiving IVR treatment. In this study, we focused on the inflammatory cytokines thought to be involved in DME and analysed its levels that could measure for systematically to identify the changes after treated with ranibizumab. A total of 26 aqueous humour samples obtained from 13 eyes of 12 patients with DME were collected. (Patients' mean age, 62.5 ± 11.9 years, mean Hb-A1c, 7.0 ± 1.3%, visual acuity, logMAR 0.47 ± 0.25, mean central foveal thickness, 570.0 ± 109.8 μm). A history of focal laser photocoagulations in two eyes was noted. A history of pan-retinal laser photocoagulations was noted for eight eyes. Intravitreal injections of ranibizumab (IVR) was performed twice in each patient. Undiluted aqueous humour samples were obtained during the IVR treatment as follows: a pre-IVR sample was taken during the initial IVR, and a second sample (post-IVR sample) was taken after completion of the second round of IVR, which was conducted 1 month after the initial IVR treatment. Individuals who had undergone previous vitreous surgery or who had already received intravitreal injections of bevacizumab were excluded from this study. Approval for this prospective study was obtained from the Institutional Review Board of our institution. Informed consent was obtained prior to study entry. A multiplex bead-based immunoassay, Luminex®100 multiplex array assay (Luminex Corporation, Austin, TX, USA), was used to measure the levels of 36 cytokines and chemokines in each aqueous humour sample. Then, nine cytokines levels were detected, which were examined between pre-IVR and post-IVR. Of note, eotaxin-1 levels in aqueous humour samples significantly decreased after IVR (p < 0.05, paired t-test, Table 1). In addition, interleukin (IL)-6 levels were also measured and analysed in samples from 12 eyes and also showed a tendency to decrease after IVR treatment. Although eotaxin-1 that was shown in this study has been reported for the characteristics in a field of age-related macular degeneration (AMD), this is the first study to show that IVR treatment reduces the levels of eotaxin-1 in the aqueous humour of DME patients. According to a previous report in AMD patients, eotaxin-1 (also known as CCL11) binds to the CCR3 receptor, and the subsequent signalling between this receptor and VEGF is particularly important to express choroidal neovascular endothelial cells in humans with AMD. Furthermore, blockade of CCR3 was more effective than VEGF neutralization in reducing choroidal neovascularization (CNV) (Kim et al. 2015). Although the pathogenesis of AMD and DME may differ, reducing eotaxin-1 levels would be expected to be a more effective function of DME therapy, due to the previously reported molecular biological interaction between VEGF and CCR3. Furthermore, IVR treatment reduces not only VEGF-A levels but also simultaneously reduces the concentration of eotaxin-1. In terms of the clinical relevance of the effects of ranibizumab treatment on the eotaxin pathway, we were unable to determine the reason for these findings. We can hypothesize that some of the effects of ranibizumab may be due to the effect on the eotaxin pathway. Additionally, significantly higher levels of IL-6 were noted in the aqueous humour of DME patients (Roh et al. 2009; Takeda et al. 2009). Funatsu et al. (2003) indicate that an IL-6-induced increase in VEGF production may contribute to DME pathology, and IL-6 may also promote an increase in vascular permeability in DME patients, in conjunction with and/or via VEGF. Considering these data, reducing the levels of IL-6 by IVR could provide an effective approach for the treatment of DME. In conclusion, although this study has a relative small sample size and further studies are warranted with a large sample size, the results of this survey suggest that this study indicates that IVR treatment can result in a significant decrease in the levels of eotaxin-1 in the aqueous humour of DME patients, with IL-6 levels following a similar trend. Furthermore, our data provide new insights into the effect of IVR on cytokine levels in the context of DME.