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Abstract
Purpose To determine whether vitreal concentrations of MCP-1, IL-6 and IL-8 are altered after vitrectomy in patients with proliferative diabetic retinopathy (PDR) and to investigate whether the altered levels of these cytokines are associated with postoperative macular oedema.
Methods Vitreous samples were collected from 36 eyes of 33 patients with PDR before pars plana vitrectomy without intraocular lens (IOL) implantation, and also from the same 36 eyes during IOL implantation surgery approximately 7 months after the initial vitrectomy. Levels of MCP-1, IL-6, IL-8 and vascular endothelial growth factor were measured by flow cytometry using cytometric bead array (CBA) technology.
Results The mean vitreous levels of MCP-1, IL-6 and IL-8 in the samples collected before vitrectomy were significantly higher in patients with PDR than in control patients (p<0.0001). The levels of MCP-1 and IL-6 in the samples collected at the time of IOL implantation were significantly higher than those collected before vitrectomy (p<0.05). In contrast, the level of IL-8 was significantly lower after vitrectomy (p<0.05). The levels of IL-6 and IL-8, but not MCP-1, in the vitreous from eyes with PDR were inversely correlated with the interval between the initial vitrectomy and the time of implantation surgery. Among the vitrectomised patients, the mean vitreous level of MCP-1 in eyes with diabetic macular oedema (DME) was significantly higher than in those without DME (p=0.028).
Conclusions The elevated levels of MCP-1 and IL-6 may indicate prolonged inflammation even after successful vitrectomy, which can cause postoperative DME.
- Vitreous
- Treatment Surgery
- Inflammation
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Introduction
Diabetic retinopathy (DR) is a leading cause of blindness in the working age population worldwide.1 The vision decrease can be caused by either proliferative DR (PDR) or diabetic macular oedema (DME). In advanced PDR, neovascularisation develops, and blindness can result from the formation of abnormal fibrovascular membranes (FVMs) with subsequent intravitreal haemorrhage and tractional retinal detachment.2 Although there are several hypotheses about the cause of DR, the specific molecular mechanisms involved remain undetermined.
Evidence has been accumulating that inflammatory processes play a significant role in the pathogenesis of DR, with several studies showing a significant correlation between inflammatory factors in both the vitreous and aqueous fluids and DR progression.3 ,4 The two major causes of impaired vision in diabetes, namely, neovascularisation (PDR)5 and increased retinal vascular permeability in eyes with DME, are accompanied by inflammation.6 ,7
Several studies including ours have demonstrated that monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF) are four major factors that are up-regulated in eyes with DME and PDR.7 ,8
Currently, pars plana vitrectomy (PPV) is the only treatment for advanced PDR.9 To determine the underlying molecular mechanisms that determine the effects of vitrectomy, we have measured the levels of angiogenesis-related factors in vitreous samples obtained from patients with PDR before PPV without IOL implantation, and also in fluid samples obtained during a second surgery for IOL implantation approximately 6 months after the initial vitrectomy.10 ,11 We found that the rate of clearance of molecules in vitrectomised eyes was different for different molecules in the vitreous. We also found a significant decrease in the intravitreal concentration of angiogenic factors including VEGF after successful vitrectomy. However, to the best of our knowledge, there is little direct evidence on how vitrectomy affects the expression of the remaining three major inflammatory factors, MCP-1, IL-6 and IL-8, that can be associated with DR.
It was recently reported that an intravitreal injection of anti-VEGF drugs may be helpful for the treatment of DME. However, one injection each month for 3 months intravitreal injections of anti-VEGF antibodies, for example, bevacizumab, in vitrectomised eyes with DME had no effect on the mean foveal thickness or mean visual acuity.12 One possible reason for the absence of an effect is the faster clearance and the subtherapeutic concentrations of bevacizumab in vitrectomised eyes.12 It is also possible that the expression profile of inflammatory factors other than VEGF may be related to the postoperative DME because VEGF is significantly reduced after vitrectomy.11 However, little is known about how altered cytokine profiles are involved in postoperative DME.
Thus, the purpose of our study was to establish whether vitrectomy alters the levels of MCP-1, IL-6 and IL-8 in the vitreous of eyes with PDR and whether the altered expression of those inflammatory factors is significantly correlated with the postoperative DME in vitrectomised eyes. To determine this, we compared the levels of MCP-1, IL-6 and IL-8 proteins in the vitreous of patients with PDR collected before vitrectomy to those collected at a second surgery performed to implant an IOL. We show that there was an increase in the intravitreal concentrations of MCP-1 and IL-6, and a significant decrease in IL-8 after the successful vitrectomy. In addition, the level of MCP-1 after vitrectomy was significantly correlated with the presence of postoperative DME in vitrectomised eyes.
Patients and methods
This study was approved by the Ethics Committees of Kyushu University Hospital and Fukuoka University Chikushi Hospital, and the surgical specimens were handled in accordance with the Declaration of Helsinki. All patients were informed of the purpose of this study and signed an informed consent form for participation beginning in June 2007. The inclusion and exclusion criteria for participation in this study are shown in online supplementary table S1.
All patients underwent a comprehensive ocular examination before each of the surgeries and periodically up to 7 months after each surgery. None of the patients underwent anti-VEGF or steroid treatment before or after the initial vitrectomy.
The DR of each patient was graded according to the modified Early Treatment Diabetic Retinopathy Study (ETDRS) retinopathy severity scale.13 In our university hospital, we have been employing a two-step surgical strategy for the treatment of patients with severe PDR. It was decided that patients with severe PDR would undergo vitrectomy without insertion of an IOL at the initial surgery, and that an IOL would be implanted only after it was confirmed that the rate of retinopathy had decreased after the vitrectomy. The indications for not inserting an IOL at the initial vitrectomy in patients with severe PDR were active retinopathy with neovascularisation on the disc, iris neovascularisation, retinal detachment, and no preoperative retinal photocoagulation.11 The IOL was implanted at least 90 days after the initial vitrectomy. The criteria for performing IOL implantation were an intraocular pressure (IOP) of <20 mm Hg, and absence of clinically significant intraocular inflammation, iris neovascularisation and vitreous haemorrhage. In addition, IOL implantation surgery was performed only in eyes without proliferative tissue.
Sample collection
Before the initial vitrectomy, samples of undiluted vitreous fluid (0.5–1.0 mL) were aspirated under standardised conditions and immediately transferred to sterile tubes.14 The samples were centrifuged for 10 min at 3000 rpm (1630×g) at 4°C, and the supernatants were divided into aliquots and stored at −70°C until analysis. After the samples were collected, lensectomy was performed on all eyes followed by a standardised PPV. During vitrectomy, the FVMs were delaminated and the posterior vitreous around the macula was removed. Then, panretinal endolaser photocoagulation (PRP) of the retina was performed up to the ora serrata. If retinal detachment was detected or developed, it was treated with an air tamponade. At the end of vitreous surgery, a hole of approximately 6 mm diameter was made at the centre of the anterior capsule, resulting in communication between the anterior chamber and vitreous cavity. This enabled us to obtain vitreous fluids from the anterior vitreous cavity at the beginning of the second surgery for IOL implantation several months later.
We collected vitreous samples from 36 eyes of 33 patients (average age 56.8±10.5 years, 22 men and 11 women) with PDR before the lensectomy and vitrectomy without IOL implantation. We also obtained 36 samples from the same eyes at the time of the secondary IOL implantation surgery. The interval between the initial vitrectomy and the secondary IOL implantation surgery was 93–722 days with a mean of 219.5±150.4 days. We determined the correlation between the concentrations of vitreal MCP-1, IL-6 and IL-8 and the interval between the initial vitrectomy and the IOL implantation surgery in the 36 eyes.
Because the concentrations of both MCP-1 and IL-6 had been reported to be significantly correlated with the presence of DME,6 ,15 we also determined whether expression of MCP-1 and IL-6 was significantly correlated with postoperative DME after successful vitrectomy. To test this, we subdivided the vitrectomised patients at the time of IOL implantation into those with DME and those without DME. DME was defined as a central foveal thickness that was 300 µm or greater by optical coherence tomography, and a retinal thickening over two or more disc areas involving some portion of the foveal avascular zone.16 ,17 None of our DME patients had undergone any treatments, including intravitreal injections of anti-VEGF, steroids, or focal photocoagulation, before the initial vitrectomy or between the initial vitrectomy and IOL implantation. The clinical characteristics of the patients are presented in online supplementary table S2.
ELISA
We measured the concentrations of MCP-1, IL-6, IL-8 and VEGF using a human Cytometric Bead Array (CBA) Kit (BD Biosciences, San Jose, California, USA) according to the manufacturer's instructions. The minimum detectable concentration was 1.3 pg/mL for MCP-1, 1.6 pg/mL for IL-6, 1.2 pg/mL for IL-8, and 4.5 pg/mL for VEGF.
Statistical analyses
Statistical analyses were performed using a commercial statistical software package (JMP, V.8.0; SAS Institute, Cary, North Carolina, USA). Categorical data were compared by Fisher's exact tests. The data were initially examined by Shapiro–Wilk tests to determine the normality of the distribution. Data that were not normally distributed were analysed by non-parametric statistics. The significance of the differences between controls and preoperative experimental groups or between controls and postoperative groups was determined by Mann–Whitney tests. The significance of the differences between the preoperative and postoperative groups was analysed by Wilcoxon matched-pairs signed-ranks tests. The correlation between MCP-1, IL-6 and IL-8 concentrations and the interval between the initial vitrectomy and the second surgery for IOL implantation was determined by the Spearman coefficient of correlation. Data are presented as means±SDs.
Results
Vitreous levels of MCP-1, IL-6 and IL-8 after successful vitrectomy
To determine the changes in the level of MCP-1, IL-6 and IL-8 after vitrectomy, we measured their concentrations in the 36 vitreous samples collected from 33 patients with PDR just before the initial vitrectomy. We also measured the concentrations in the 36 samples from the same eyes collected before the secondary surgery for IOL implantation.
The PDR grade before vitrectomy was level 71 in 14 eyes, level 75 in seven eyes, level 81 in six eyes, and level 85 in nine eyes using the ETDRS scale (see online supplementary figure S1A). At the time of the second surgery for IOL implantation, the PDR was improved by three levels or more in all 36 eyes: to level 35 in one eye, level 43 in 14 eyes, level 47 in 14 eyes, level 53 in five eyes, level 61 in one eye and level 65 in one eye.
The mean visual acuity was 0.97 logarithm of the minimum angle of resolution (logMAR) units at baseline, which improved significantly to 0.34 logMAR units at the time of the surgery for IOL implantation (p<0.0001; see online supplementary figure S1B). Twenty-eight (77.8%) of the eyes had an improvement of two or more lines in visual acuity after surgery and four (11.1%) were unchanged. Four eyes (11.1%) had a worse visual acuity: two eyes with neovascular glaucoma that was present before the initial vitrectomy, and two eyes that had hard exudates adjacent to the fovea. Ultimately, 33 eyes (91.7%) had a visual acuity of 20/200 or better.
Consistent with the previous report,5 the mean concentration of MCP-1, IL-6 and IL-8 in the vitreous was significantly higher in the 36 vitreous samples collected from 33 patients with PDR (929.5±69.65 pg/mL, 23.59±3.05 pg/mL and 49.64±7.01 pg/mL, respectively) than in the vitreous of the control patients (120.9±11.98 pg/mL (figure 1A), 2.71±1.43 pg/mL (figure 2A) and 1.15±0.45 pg/mL (figure 3A), respectively; p<0.0001).
MCP-1 concentrations in the vitreous samples of non-diabetic macular hole (MH) patients, proliferative diabetic retinopathy (PDR) patients, and vitrectomised PDR patients at the time of intraocular lens (IOL) implantation after an earlier vitrectomy (PDR after PPV). (A) The MCP-1 level is significantly higher in eyes with PDR than in the MH eyes at the time of the initial vitrectomy (*p<0.0001). The MCP-1 level at the time of the secondary surgery for IOL implantation was also significantly higher than the level in the vitreous collected at the initial vitrectomy (**p=0.042). PPV, pars plana vitrectomy. (B) Scatter plots comparing MCP-1 levels in eyes with PDR at the time of the initial vitrectomy (preoperative) to those at the time of the secondary surgery for IOL implantation (postoperative).
IL-6 concentrations in the vitreous samples of non-diabetic macular hole (MH) patients, proliferative diabetic retinopathy (PDR) patients, and vitrectomised PDR patients at the time of intraocular lens (IOL) implantation after an earlier vitrectomy (PDR after PPV). (A) The IL-6 level is significantly higher in eyes with PDR at the time of initial vitrectomy than in MH patients (*p<0.0001). The IL-6 level at the time of the secondary surgery for IOL implantation was also significantly higher than the level in the vitreous collected at the initial vitrectomy (**p=0.039). PPV, pars plana vitrectomy. (B) Scatter plot comparing IL-6 levels in eyes with PDR at the time of initial vitrectomy (preoperative) to those at the time of the secondary surgery for IOL implantation (postoperative).
IL-8 concentrations in the vitreous samples of non-diabetic macular hole (MH) patients, proliferative diabetic retinopathy (PDR) patients, and vitrectomised PDR patients at the time of intraocular lens (IOL) implantation after an earlier vitrectomy (PDR after PPV). (A) The IL-8 level is significantly higher in eyes with PDR at the time of initial vitrectomy than in MH patients (*p<0.0001). The IL-8 level at the time of the secondary surgery for IOL implantation is also higher than in the controls (*p<0.0001), but significantly lower than that in the vitreous samples at the time of initial vitrectomy (*p=0.038). PPV, pars plana vitrectomy. (B) Scatter plot comparing IL-8 levels in eyes with PDR at the time of initial vitrectomy (preoperative) to those at the time of the secondary surgery for IOL implantation (postoperative).
At the time of IOL implantation, the MCP-1 and IL-6 levels were significantly higher (1189.6±103.33 pg/mL and 90.95±32.66 pg/mL, respectively) than the levels in the vitreous collected at the initial vitrectomy (929.5±69.65 pg/mL (p=0.042) and 90.95±32.66 pg/mL (p=0.039), respectively). Compared to the level at the time of initial vitrectomy, the level of MCP-1 was lower in 11 eyes and higher in 25 eyes (figure 1B) at the time of IOL implantation. The IL-6 level was lower in 13 and higher in 23 of 36 eyes (figure 2B) at the time of IOL implantation.
In contrast, the level of IL-8 at the time of IOL implantation was significantly lower than the level in the vitreous collected at the initial vitrectomy (p=0.038), although the IL-8 level was still significantly higher (30.19±2.39 pg/mL; p<0.0001) than that in the control patients. Compared to the level at the time of initial vitrectomy, the IL-8 level was lower in 22 eyes and higher in 14 eyes at the time of IOL implantation surgery (figure 3B). No significant correlation between the IL-8 level and the improvement in the ETDRS retinopathy severity scale was detected (data not shown).
The MCP-1 concentration at IOL implantation surgery was not correlated significantly with the interval between the initial vitrectomy and IOL implantation surgery (p=0.207; figure 4A). In contrast, the concentrations of both IL-6 and IL-8 were significantly and inversely correlated with the interval between the first vitrectomy and the surgery for IOL implantation (r=−0.431, p=0.009 and r=−0.356, p=0.036, respectively; figure 4B,C).
Correlation between vitreous MCP-1 (A), IL-6 (B) and IL-8 (C) levels at the time of intraocular lens (IOL) implantation surgery after vitrectomy with days after initial vitrectomy in 36 eyes of 33 patients with proliferative diabetic retinopathy (PDR). The MCP-1 level is not significantly correlated with the days after the initial vitrectomy (p=0.207). In contrast, there is a significant inverse correlation between IL-6 and IL-8 concentration and days after the initial vitrectomy (r=−0.432, p=0.009 and r=−0.356, p=0.036, respectively). PPV, pars plana vitrectomy.
Correlation between the presence of postoperative DME and vitreous levels of MCP-1, IL-6, IL-8 and VEGF after successful vitrectomy
For the 36 eyes with PDR, the vitreous level of MCP-1 at the time of IOL implantation surgery was 1473.2±240.9 pg/mL in the 12 eyes with DME and 1047.8±83.7 pg/mL in the 24 eyes without DME. This difference was statistically significant (p=0.028; figure 5A). In contrast, IL-6, IL-8 and VEGF concentrations in the 12 eyes with DME (159.9±87.4 pg/mL, 28.24±3.48 pg/mL and 44.39±11.67 pg/mL, respectively) were not significantly different from those in 24 patients without DME (56.50±18.44 pg/mL, 33.52±4.11 pg/mL and 61.74±19.47 pg/mL, respectively; figure 5B–D).
Intravitreous levels of MCP-1, IL-6, IL-8 and vascular endothelial growth factor (VEGF) at the time of intraocular lens (IOL) implantation surgery and their relationship with the presence or absence of diabetic macular edema (DME). (A) Intravitreous level of MCP-1 in eyes with and without DME (*p<0.05). (B) Intravitreous level of IL-6 in eyes with and without DME. N.S., not significant. (C) Intravitreous level of IL-8 according to the presence or absence of DME. N.S., not significant. (D) Intravitreous level of VEGF according to the presence or absence of DME. N.S., not significant.
Discussion
We showed earlier that the vitreal levels of VEGF, erythropoietin, angiopoietin-2 and hepatocyte growth factor in patients with PDR were significantly reduced after an initial vitrectomy.10 ,11 In contrast, the levels of endostatin, TIMP-1, TIMP-2 and total protein were found not to be decreased significantly after an initial vitrectomy. In the current study, the potent proinflammatory factors MCP-1 and IL-6 were significantly increased at approximately 7 months after vitrectomy when clinically apparent inflammation was absent (figures 1 and 2). In contrast, IL-8 was significantly reduced after the initial vitrectomy (figure 3). These findings indicate that successful vitrectomy can cause a shift toward a more proinflammatory microenvironment by changing the cytokine expression profile. However, this contradicts general clinical findings that DME in many PDR patients improves after vitrectomy even without treatment for the DME. This discrepancy is probably due to use of the procedures associated with vitrectomy rather than the more proinflammatory state that is induced by vitrectomy. An example of this is the inflammatory condition found after photocoagulation. Aphakia after vitrectomy is not a physiological state, and might cause the shift in the cytokine profile.
Although we found that the concentrations of IL-6 and IL-8 protein were reduced in a time-dependent manner (figure 4B,C), it is not likely that the kinetics of IL-6 and IL-8 decreases linearly after vitrectomy. This is because the level of IL-6 in 25 eyes and the level of IL-8 in 14 eyes had already increased at the time of IOL implantation surgery (figures 2B and 3B). Because vitrectomy is an invasive procedure that leads to inflammation and breakdown of the blood–ocular barrier which may last for a few months after surgery,11 it is reasonable that the increase in the IL-6 and IL-8 levels was more likely due to the inflammation induced by the procedures associated with vitrectomy and that these levels then gradually decreased thereafter.
In contrast, no significant correlation was found between the level of MCP-1 and the interval between the initial vitrectomy and IOL implantation surgery (figure 4A). This is probably because the level of MCP-1 at the time of IOL implantation surgery was more susceptible to variations in the invasiveness of the vitrectomy rather than the time-dependent increased clearance effect of vitrectomy. This is in agreement with a previous report that showed an elevation in MCP-1 even after a mean postoperative period of 17 months when clinical examinations showed no inflammatory responses in the eye.18 Therefore, MCP-1 may be the most important modulator after vitrectomy among the three major proinflammatory cytokines examined. In support of this, the mean vitreous level of only MCP-1 was significantly higher in vitrectomised eyes with DME than in eyes without DME (figure 5A). Our findings suggest that MCP-1 can be a factor contributing to the postoperative DME in vitrectomised eyes. MCP-1 is a chemokine that recruits monocytes/macrophages into tissues.19 Moreover, the recruitment of monocytes/macrophages to vessel walls promotes vascular permeability potentiating the DME.20
Similar to MCP-1, the level of IL-6 is also increased after surgical procedures (figure 2).21 Interestingly, the four eyes with the highest IL-6 at the time of IOL implantation all had retinal detachment at the time of initial vitrectomy. Therefore, it may be possible that IL-6 expression is particularly enhanced by tissue repair processes during retinal re-attachment in eyes with DR. IL-6 is a cytokine that functions throughout the inflammatory cascade and can cause an increase in vascular permeability.22 In our study, there was a non-significant trend towards a positive association between IL-6 concentration and the presence or absence of DME at the time of IOL implant surgery (figure 5B). We assume that the lack of a significant correlation was due to the small sample size, and further studies with a larger sample size are needed to demonstrate a significant correlation between IL-6 level and the presence of DME after vitrectomy.
We found earlier that IL-8, a pro-inflammatory and angiogenic cytokine, is synthesised by endothelial and glial cells in ischaemic retinas.8 A number of studies have demonstrated that IL-8 appears to also play a role in the development of DME.7 ,23 In addition, the expression of IL-8 in the retina and vitreous cannot be reduced by intravitreal steroids or anti-VEGF treatment.7 In our study, we found that vitrectomy is an effective way to reduce the level of vitreous IL-8 similar to VEGF (figures 3 and 4C). This is probably because vitrectomy can increase oxygenation of the ischaemic retina.24
In contrast to the weak effects of anti-VEGF treatment on DME in vitrectomised eyes,12 treatment with intravitreal implants of dexamethasone led to significant improvements in both vision and vascular leakage from the DME in vitrectomised eyes.25 Because elevated levels of MCP-1 and IL-6 were significantly reduced following treatment with intravitreal steroids in association with a reduction in DME,16 the more favourable effects of dexamethasone implants compared to anti-VEGF treatment were most likely mediated by reduced levels of MCP-1 and IL-6. Consequently, a more thorough blocking of MCP-1 and IL-6 by developing specific molecular targeting therapies such as an antibody against each molecule, may reduce the percentage of eyes that develop DME in difficult-to-treat vitrectomised eyes.
In conclusion, we found a significant increase in the intravitreal concentrations of MCP-1 and IL-6, and a significant decrease in IL-8 after successful vitrectomy in patients with PDR. In addition, the level of MCP-1 after vitrectomy was significantly correlated with the presence of postoperative DME in vitrectomised eyes. These findings indicate that the presence of prolonged inflammation in the vitreous even after successful vitrectomy may be the cause of postoperative DME.
Acknowledgments
We thank Masayo Eto who provided excellent technical help.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
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Footnotes
Contributors SY was responsible for study concept and design, data collection and analysis, and writing the manuscript. YKu, YKo, YZ, TN, MY, TT and KI contributed to data analysis. RA, SN, YS, YO and TK contributed to data collection. TI contributed to review of the manuscript and approved publication of the manuscript.
Funding This work was supported in part by JSPS KAKENHI grant numbers 24249083, 26293374 and 26670757 and Takeda Science Foundation.
Competing interests None.
Ethics approval The Ethics Committees of Kyushu University Hospital (24-164) and Fukuoka University Chikushi Hospital (R08-019) approved this study.
Provenance and peer review Not commissioned; externally peer reviewed.
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