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. 2015 Jul 14;181(2):338–342. doi: 10.1111/cei.12627

Chemokine CXCL-1: activity in the vitreous during proliferative vitreoretinopathy

C Symeonidis *, S Androudi †,✉, I Georgalas ‡, A Tzamalis *, N Chalvatzis *, T Rotsos ‡, E Souliou §, E Diza , S A Dimitrakos *
PMCID: PMC4516449  PMID: 25766782

Abstract

The aim of this study was to investigate CXCL-1 chemokine levels in the vitreous during rhegmatogenous retinal detachment (RRD) with and without proliferative vitreoretinopathy (PVR) and identify possible correlations with clinical parameters (extent and duration or RRD and PVR grade). Vitreous samples from patients with primary RRD with or without PVR were collected and assayed using a double antibody enzyme-linked immunosorbent assay (ELISA). Eleven vitreous samples from organ donors were employed as a control group. CXCL-1 levels were measured in 35 vitreous samples from 35 RRD patients. Mean CXCL-1 levels (64·82 ± 6·47 pg/ml) were significantly higher (P = 0·048) compared to controls. There was a significant positive correlation between CXCL-1 levels and the extent of the detachment (r = 0·794, P = 0·006). Peak CXCL-1 levels coincided with 3+ quadrant RRD, an interim of 29–60 days’ duration and PVR grade B. Increased CXCL-1 levels may be indicative of mild inflammation in the detached retina and the adjacent vitreous. The results of the present study may provide novel insight into the complex interactions taking place during the early and late stages of RRD complicated by PVR.

Keywords: CXCL-1, GRO-α, rhegmatogenous retinal detachment, proliferative vitreoretinopathy, subretinal fluid

Introduction

Leucocyte extravasation and chemotaxis constitute significant stages of the wound-healing process. In these pathological circumstances, the involvement of chemokines, a family of 8–10 kDa proteins, appears to be prominent 1. Chemokines may also be implicated in leucocyte attraction, cell activation, haematopoiesis, angiogenesis and angiostasis 2. Two cysteine residues and the potential interjection of one or several amino acids act as the basis of chemokine categorization in the CC, CXC, XC, CX3C subfamilies. A human analogue of the mouse keratinocyte-derived chemokine, growth related oncogene (GRO)-α, is a member of the CXC subfamily of chemokines and is also designated as CXCL-1 according to the aforementioned classification 3. CXCL-1 is a potent neutrophil chemoattractant as well as an inducer of acute inflammatory reaction 4.

Rhegmatogenous retinal detachment (RRD) is a potentially sight-threatening clinical entity initiated by a retinal break 5. In several cases of delayed surgical management, proliferative vitreoretinopathy (PVR) may be observed. This pathological wound-healing is responsible, to a great extent, for impaired retinal and therefore failure of its anatomical and functional restoration 6.

The aim of this study was the investigation of CXCL-1 activity in the vitreous during RRD with or without PVR complication. Moreover, potential correlations of CXCL-1 activity with clinical parameters such as duration and extent of RRD and PVR grade are also investigated.

Materials and methods

Patient population

Patients (age requirement 18 years or older to be eligible for inclusion in this study) with primary RRD possibly complicated by PVR were included. Patients suffering from giant breaks (>3 clock hours) and systemic and/or intra-ocular lymphoma were excluded from this study. Patients with uveitis, proliferative diabetic retinopathy, intra-ocular surgery during the previous 6 months, history of recent (<6 months) treatment with oral steroids as well as intra-ocular steroid or anti-vascular endothelial growth factors injections within 3 months prior to RRD onset were excluded. Samples contaminated with blood during collection were also excluded.

Preoperatively, a thorough personal history was recorded for each subject enrolled into the study. The history included: an estimated onset of RRD based on the onset of patient’s symptoms (categorized in six groups: <3 days, 4–7 days, 8–14 days, 15–28 days and 29–60 days) and extent [recorded in clock hours (1–12) and categorized in quadrants (1–4)]. The Retina Society Terminology Committee classification was used in order to grade PVR 7. The Institutional Review Board of the ‘Papageorgiou’ General Hospital approved the study protocol. Informed consent was obtained by all patients included in this study.

Sample collection

One qualified and experienced vitreoretinal surgeon completed the required surgical procedures (SD). At the beginning of the vitrectomy procedure an undiluted vitreous sample was collected into a sterile syringe. The sample was centrifuged promptly at 5000 g for 15 min, at a temperature of 15 °C. Sterile Eppendorf tubes were used for supernatant storage in quantities up to 200 μl (depending on availability), followed by storage at −70 °C. A double antibody enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA) was used in order to measure CXCL-1 concentrations in pg/ml (sensitivity: 10 pg/ml).

Human organ-donor eyes were used as a control group according to relevant studies 8. Data obtained from organ-donor vitreous have been demonstrated to provide acceptable results, according to similar studies. Eleven vitreous samples were harvested from eyes within a period of 3 h post-mortem, in order to eliminate the risk of biochemical changes of the vitreous after death 8. The donors (seven men, four women; mean age 58·3 years, range: 19–71 years) had no history of ocular pathology or other systemic disorders that could lead to alteration of their vitreous composition.

Statistical analysis

In this study, CXCL-1 concentrations are reported in pg/ml (mean ± standard error of the mean). Statistical analysis was performed with the use of Spearman’s rank correlation test and the Mann–Whitney non-parametric test. Statistically significant levels were at P ≤ 0·05. The spss version 12·0 statistical package (SPSS Inc., Chicago, IL, USA) was used for the aforementioned statistical analysis. Power analysis was performed using the GPower version 3·1 ·7 statistical software (Heinrich-Heine Universität, Dusseldorf, Germany).

Results

In this study, 35 vitreous samples from 35 eyes of 35 consecutive patients diagnosed with RRD were collected. Our study population consisted of 27 male (77·1%) and eight female (22·9%) patients with a mean patient age of 62·51 years (range = 30–91). Mean CXCL-1 levels in the patient group were 64·82 ± 6·47 pg/ml, while CXCL-1 levels were 41·63 ± 9·27 pg/ml in the control group. Mean CXCL-1 levels in the patient group were elevated significantly (P = 0·048) compared to the respective CXCL-1 levers in the control group. Patients were also categorized according to PVR grade: eight patients with no PVR, four patients with PVR grade A, 13 with PVR grade B and 10 with PVR grade C were recruited into the present study. For an effect size of 0·80, a statistical power of 0·619 was determined (independent-samples t-test) in this study.

A significant correlation between RRD extent and duration (r = 0·577, P = 0·001) was observed using correlation analysis. Moreover, there were significant correlations observed between RRD extent and PVR (r = 0·366, P = 0·036), as well as RRD duration and PVR (r = 0·588, P = 0·001).

Regarding patient age, no significant correlation was observed with CXCL-1 levels (P = 0·763). With regard to gender, statistical analysis revealed no statistically significant differences between male and female patients (P = 0·884).

Regarding clinical parameters, there was a significant positive correlation between CXCL-1 levels and the extent of the detachment (r = 0·794, P = 0·006). According to graphical representation of CXCL-1 levels in relation to RRD extent (recorded in quadrants), CXCL-1 levels in the vitreous increased with the extent of the detachment and peaked with RRD cases involving 3+ quadrants (Fig. 1). There was no difference between CXCL-1 levels that coincided with total RRD and CXCL-1 levels that coincided with total (4-quadrant) RRD.

Figure 1.

Figure 1

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Graphical representation of mean CXCL-1 levels as per extent and duration of the detachment and proliferative vitreoretinopathy in the vitreous of patients with rhegmatogenous retinal detachment.

Concerning RRD duration, no significant correlation with CXCL-1 levels (P = 0·650) was observed. Moreover, graphical representation revealed that CXCL-1 levels in the vitreous increased with RRD duration and peaked during days 29–60 (Fig. 1).

Regarding PVR, there was no significant correlation with PVR grade (P = 0·437). According to graphical representation, CXCL-1 levels in the vitreous increased with PVR grade (Fig. 1).

Moreover, regression analysis revealed a significant positive correlation between CXCL-1 levels and RRD extent (R = 0·718, P = 0·019). No significant correlations were observed between CXCL-1 and RRD duration (P = 0·605) or PVR grade (P = 0·637).

Discussion

Primary RRD appears to be a pathological environment characterized by abundant stimuli. In this context, chemokine levels have been shown to be increased compared to controls pre- as well as postoperatively, an observation suggesting local production 9,10.

The activity of chemokine CXCL-1/GRO-α as a powerful chemoattractant in ocular tissues and elsewhere have been reported previously 11,12. In the present study, CXCL-1 was measured in the vitreous in the context of RRD. To our knowledge, this is the first study reporting CXCL-1 levels in the vitreous of RRD patients with and without PVR. Retinal pigment epithelial (RPE) cells have been shown to produce CXCL-1 as a result of PVR-related mediators in addition to being components of the RRD milieu and PVR membranes 13. According to the results of the present study, CXCL-1 levels were elevated compared to controls, an observation suggesting local production in the detached retina and the adjacent vitreous during these pathological circumstances. This statistically significant increase may be a result of low-grade inflammation. In the present study, vitreous from organ donor eyes was used as control. The use of laboratory data obtained from organ-donor vitreous is regarded widely as adequate when compared to vitreous obtained from a number of vitreoretinal diseases in similar studies (e.g. macular puckers) 14,15.

Correlations with clinical parameters

Regarding RRD pathogenesis, a number of models have been proposed. A number of models focus on inflammation and cells associated with this process, while several other models focus on growth factors and cytokines detected in the vitreous during RRD 16. The observed localized inflammation appears not to be confined to the detached retina but to the adjacent non-detached retina as well, and is probably the result of a process including inflammation, immune responses and fibrinolysis set about by photoreceptor destruction 17. In this context, molecules [e.g. interleukin (IL)-6] associated with inflammation have been reported previously to correlate with clinical parameters, such as extent, thus rendering inflammation clinically important 18.

According to the results of this study, correlation analysis revealed a significant positive correlation between RRD extent and duration. Moreover, there were significant correlations between RRD extent and PVR as well as between RRD duration and PVR. The findings of the present study are consistent with previous models of PVR development 19. A detached retina of greater extent may lead to elevated quantity of RPE cells and fibroblasts in the adjacent vitreous, thus increasing the probability of PVR. An RRD of prolonged duration may also be complicated with PVR by facilitating RPE cell dedifferentiation. PVR membranes are characterized by greater density as well as number of cells. The subsequently observed cellular proliferation may, in all probability, contribute significantly to the rapid expansion of PVR membranes 19. This plethora of significant correlations between all investigated clinical parameters may be indicative of vitreous suitability for the assessment of CXCL-1 involvement in RRD and PVR.

Age

With respect to age, there was no significant correlation observed with CXCL-1 levels. This finding is consistent with a similar study reporting no significant influence of age on chemokine expression after spinal cord injury 16. It appears that there were no detectable changes in the inflammatory response brought about by age in the context of RRD with or without PVR.

Gender

With respect to gender, no statistically significant difference was revealed between male and female patients. According to a relevant study, there were elevated CXCL-1 levels observed in the serum of patients with spinal cord injury, but there was difference regarding gender 16. Consequently, the finding of the present study is consistent with previous studies on the role of gender in chemokine activity in the context of RRD.

CXCL-1-extent

In this study, there was a significant correlation between CXCL-1 levels and RRD extent. Furthermore, peak CXCL-1 levels coincided with 3+ quadrant RRD extent, while there was no difference with CXCL-1 levels that coincided with total RRD. A limited number of studies have reported CXCL-1 activity in the eye in the context of inflammation 13. Vitreous from an RRD of significant extent is characterized by a significant rise in interleukin as well as matrix metalloproteinase activity which, in turn, act as stimuli for CXCL-1 production 14. This finding may be an indication of CXCL-1 contribution to RRD pathophysiology. Moreover, regression analysis revealed a significant link between CXCL-1 and RRD extent, a finding also potentially indicative of a role for CXCL-1 in RRD pathophysiology. Further studies could investigate the potential predictive value of this particular chemokine for RRD extent.

CXCL-1-duration of RRD

With regard to RRD duration, there was no significant correlation of this clinical parameter with CXCL-1 levels (although we have to acknowledge that the accurate estimate of the onset of RRD based on the reported patient’s symptoms can be extremely complicated and highly variable). Moreover, graphical representation depicted that CXCL-1 levels coincided with a 29–60-day detachment duration. This finding may indicate that prolonged RRD duration resulted in increased chemokine activity in the vitreous in the pathological context of RRD. Nevertheless, interactions between various chemokines appear to be complex: further studies are required in order to clarify the level of CXCL-1 implication in the wound healing process during RRD.

CXCL-1- PVR

A number of molecules have been reported to be elevated during PVR and identified subsequently as potential therapeutic targets 20. Potential correlations between concentrations and PVR presence but not grade were investigated. Moreover, the control vitreous samples employed for this study were obtained from macular puckers, a clinical entity that may be considered as a proliferative pathological entity. According to the results of this study, CXCL-1 levels were increased during PVR but did not correlate significantly with PVR grade. This observation is in agreement with previous studies reporting chemokine involvement in wound-healing and inflammation 21. The observation that CXCL-1 levels decreased during PVR grade C may also account for the lack of a statistically significant correlation between CXCL-1 and PVR grade. Graphical representation of CXCL-1 levels with PVR grade revealed a peak that coincided with grade B, a relatively uncomplicated grade. As an increase was observed between CXCL-1 levels in RRD cases not complicated with PVR and RRD cases with PVR grade B, this finding may be an indication of inflammation adjacent to the detached retina that could be viewed as result of PVR. The results of the present study suggest that CXCL-1 may be a feasible therapeutic target in the context of PVR.

In this study, a significant increase in CXCL-1 levels in patients with RRD was observed, a finding in agreement with the relevant literature on chemokine involvement in inflammation and wound-healing 22,23. A significant positive correlation between CXCL-1 levels and the extent of the detachment was recorded. According to a graphical representation, peak CXCL-1 levels coincided with a 3+ quadrant extent, a 29–60-day duration and PVR grade B. A significant positive correlation between CXCL-1 levels and RRD extent was also observed using regression analysis. Increased CXCL-1 levels may be indicative of mild inflammation in the detached retina and the adjacent vitreous. The results of the present study may provide novel insight in the complex interactions taking place during RRD as well as PVR.

Disclosure

The authors have no financial interests to disclose.

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