Comprehensive analysis of inflammatory immune mediators in vitreoretinal diseases

Abstract

Inflammation affects the formation and the progression of various vitreoretinal diseases. We performed a comprehensive analysis of inflammatory immune mediators in the vitreous fluids from total of 345 patients with diabetic macular edema (DME, n = 92), proliferative diabetic retinopathy (PDR, n = 147), branch retinal vein occlusion (BRVO, n = 30), central retinal vein occlusion (CRVO, n = 13) and rhegmatogenous retinal detachment (RRD, n = 63). As a control, we selected a total of 83 patients with either idiopathic macular hole (MH) or idiopathic epiretinal membrane (ERM) that were free of major pathogenic intraocular changes, such as ischemic retina and proliferative membranes. The concentrations of 20 soluble factors (nine cytokines, six chemokines, and five growth factors) were measured simultaneously by multiplex bead analysis system. Out of 20 soluble factors, three factors: interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) were significantly elevated in all groups of vitreoretinal diseases (DME, PDR, BRVO, CRVO, and RRD) compared with control group. According to the correlation analysis in the individual patient's level, these three factors that were simultaneously increased, did not show any independent upregulation in all the examined diseases. Vascular endothelial growth factor (VEGF) was significantly elevated in patients with PDR and CRVO. In PDR patients, the elevation of VEGF was significantly correlated with the three factors: IL-6, IL-8, and MCP-1, while no significant correlation was observed in CRVO patients. In conclusion, multiplex bead system enabled a comprehensive soluble factor analysis in vitreous fluid derived from variety of patients. Major three factors: IL-6, IL-8, and MCP-1 were strongly correlated with each other indicating a common pathway involved in inflammation process in vitreoretinal diseases.

Figure 1. Detection of soluble factors in vitreous fluids from patients.

(A) IL-6, (B) IL-8, (C) MCP-1, and (D) VEGF levels in vitreous fluid of control patients and patients with DME, PDR, BRVO, CRVO, and RRD. The ordinate showed the concentrations of soluble factors in the log scale, bars represent the mean value of each group. * P<0.05, N.S.: not significant.

Figure 2. IL-6, IL-8 and MCP-1 were mutually increased in all examined vitreoretinal diseases.

As an example, Spearman's correlation analysis of DME are shown (n = 92). In each disease, total of three combinations (IL-6/IL-8, IL-6/MCP-1, and IL-8/MCP-1) were created, and calculated p- and r-values which indicating the accuracy of correlation.

Figure 3. Summary of correlation analysis.

The combination of two factors was calculated for each individual disease and listed in the ordinate.

Figure 4. VEGF independently contribute to the pathogenic process of PDR and CRVO.

Spearman's correlation analysis between VEGF and IL-6/IL-8/MCP-1 in patients with PDR (n = 147) or CRVO (n = 13) are shown. In each disease, total of three combinations (VEGF/IL-6, VEGF/IL-8, and VEGF/MCP-1) were analyzed, and calculated p- and r-values which indicating the accuracy of correlation.

Figure 5. Serum IL-6 and VEGF concentrations are not significantly related with vitreous concentrations.

Shown are comparison of (A) IL-6, and (B) VEGF levels in vitreous and in serum of patients with control (n = 53), PDR (n = 66) and CRVO (n = 8). The ordinate showed the concentrations of soluble factors in the log scale, bars represent the mean value of each group. Bars represent the mean value of each group. * P<0.01, N.S.: not significant.

Figure 6. Possible contribution of major four factors: IL-6, IL-8, MCP-1 and VEGF in vitreoretinal diseases.

A scheme illustrates the relations between the different mediators in samples from the different vitreoretinal diseases: IL-6, IL-8, and MCP-1 in the vitreous cavity can increase vascular permeability that causes DME, but an additional retinal ischemia led to increased VEGF production that play an important role in the progression of DME to PDR. At the same time, high VEGF levels may be initially produced by the sudden profound retinal injury, and then induced major three factors, frequently resulted in rubeosis iriditis.