Clinical immunology

Cytokines in the subretinal fluid of patients with rhegmatogenous retinal detachment

Introduction


Retinal detachment is a serious ophthalmic disorder, whose aetiology has not been completely explained. The detachment of over one-month duration shows complications with proliferative vitreoretinopathy. Recent studies have demonstrated that cytokines are involved in this process. The presence of interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), tumour necrosis factor alpha (TNF-α), interferon gamma (IFN-γ) and monocyte chemotactic protein-1 (MCP-1) has been revealed in the vitreous and within membranes of detached retina in patients with PVR [1-6].
Cells which infiltrate these eye structures and breakdown of the blood-retinal barrier are the likely sources of these cytokines.
Our previous studies revealed the presence of IL-6 and such cells as lymphocytes, macrophages, mastocytes and retinal pigment epithelial cells in the subretinal fluid (SRF) [7].
In the present study the levels of IL-8, IL-10, IL-I3, TNF-alpha and IFN-gamma were evaluated in the subretinal fluid of patients operated on for rhegmatogenous retinal detachment of varied duration.


Material and methods


The study included 46 patients – 18 men and 28 women aged 10-85 with rhegmatogenous retinal detachment. Full ophthalmic examination revealed aphakia in 3 eyes, myopia in 15, hypermetropia in 4 and emmetropia in the remaining patients. In 11 eyes microcystic degeneration-type changes were found on the eye fundus periphery.
The time range of detachment was up to 2 weeks in 12 patients, 3-4 weeks in 13 cases, 5-8 weeks in 11 and over 2 months in 10 patients.
Visual acuity prior to surgery was defined as light perception with normal localisation in 26 patients, less than 5/50 in 12 and 5/50 – 5/12 in 8 eyes.
Retinal detachment referred to 2 fundus quadrants in 22 cases, 3 quadrants in 17 patients and 4 quadrants in 7 patients.
A single retinal hole was localised in the upper part of the eye fundus in 25 patients, in the lower part in 16 cases; two holes were found in the lower part of the retina in 5 cases.
Type A proliferative vitreoretinopathy was observed in 11 cases, type B in 11 and type C in 10 patients.
Aspirates of subretinal fluid, 1.0-2.0 ml each, were obtained during surgery performed in general anaesthesia. Samples contaminated with blood were excluded from the study. In 36 patients the operative technique consisted in the episcleral buckling, in 4 cases a meridonal-epidural implant was added. 10 patients which had PVR grade C treated by pars plana vitrectomy.
The immunoenzymatic method was applied to determine the concentrations of IL-8, IL-10, IL-13, TNF-alpha and IFN-gamma in SRF. Quantikine kits (Research and Diagnostic System, Minneapolis and Endogen, Cambridge, USA) were used. Assays were carried out following the manufacturer’s procedure, with proper interleukin standards.
The results obtained were subjected to statistical analysis with t-Student test (significant values at p<0.05).


Results


The presence of IL-8, IL-10, IL-13, TNF-alpha and IFN-gamma was demonstrated in all the subretinal fluid samples examined.
Correlations between the levels of IL-8, IL-10, IL-13, TNF-alpha, IFN-gamma and detachment time have been shown in Table 1 (Table 1).
The level of IL-8 in the subretinal fluid of patients with 1-8 week detachment ranged from 585±75 pg/ml to 648±200 pg/ml, being 516±164 pg/ml in the detachment of longer duration. The analysis of the correlation between IL-8 level and detachment time revealed statistically significant differences between 5-8 week and over 2 month detachment (p=0.012).
The concentrations of TNF-alpha in the subretinal fluid of patients with detachment time shorter than 2 months ranged from 185±75 pg/ml to 250±128 pg/ml, showing a falling tendency in the samples obtained from patients with detachment of over 8 weeks’ duration (173±65 pg/ml). Statistically significant differences were found between detachment duration of 5-8 weeks and that of over 2 months (p<0.05).
The concentrations of IFN-gamma in the subretinal fluid of patients with 1-8 week detachment ranged from 65±25 pg/ml to 72±13 pg/ml, showing a slight decrease after 8 weeks of detachment to 56±10 pg/ml. Differences between the respective periods of detachment were statistically insignificant.
The activity of IL-10 was the highest in the subretinal fluid of patients with 3-4 week detachment (108±34 pg/ml), showing a decrease to 53±28 pg/ml - 56±31 pg/ml in detachment longer than 4 weeks. Statistically significant differences were found between detachments lasting 1-2 weeks, 5-8 weeks, over 2 months and that of 3-4 weeks’ duration (p<0.001).
The concentrations of IL-13 in the subretinal fluid ranged from 390±92 pg/ml to 424±79 pg/ml. No correlation was observed between IL-13 concentration and time of retinal detachment.
No significant differences were found between the concentrations of the cytokines examined and detachment extent.
The concentration of IL-8 ranged from 559±184 to 613±199, the level of IL-10 was between 62±21 and 68±29 pg/ml, and of IL-13 between 390±70 and 420±98 pg/ml. The activity of TNF-alpha was found to range from 180±76 to 238±90 pg/ml, while the activity of IFN-gamma from 62±15 to 68±21 pg/ml.
The results are presented in Table 2 (Table 2).


Discussion


Our study revealed the presence of IL-8, IL-10, IL-13, TNF-alpha and IGN-gamma in the subretinal fluid of patients with rhegmatogenous retinal detachment. The concentration of IL-8 was the highest. The presence of this cytokine has been revealed by Elner in the material obtained from patients with proliferative vitreoretinopathy [8]. Its role in PVR, however, has not been fully elucidated yet. Il-8 is a chemotactic factor for neutrophils, and TNF-alpha present in the subretinal fluid induces IL-8 and MCP-1 production [9].
Retinal pigment epithelial cells, especially after IL-1 beta, TNF alpha or LPS stimulation, as well as local macrophages are the likely sources of IL-8. Cells expressing mRNA for IL-8 and TNF-alpha in the vitreous and subretinal fluids have been found by El-Ghrrably in most patients with PVR [10].
Hackett et al. [11] have revealed that retinal epithelial cells proliferate and migrate depending on the extent and time of detachment, and become morphologically and functionally similar to macrophages. We found the highest concentrations of IL-8 and TNF-alpha in retinal detachment shorter than 2 months.
Studies evaluating the concentrations of TNF-alpha and IFN-gamma in the vitreous of patients with PVR-complicated detachment and with uncomplicated detachment have found low TNF-alpha concentrations in more cases with PVR than without. The authors believe that the inflammation-activating cytokines affect PVR. These cytokines may be locally produced by cells infiltrating the epiretinal membranes [12].
Slepova et al. [13] have shown the presence of TNF-alpha in the subretinal fluid of patients with panophthalmia. Its concentration correlated with severity of the pathologic process. The level of IFN-gamma was the lowest in the subretinal fluid. Levels of TNF-alpha and IFN-gamma were higher in patients with diabetic retinopathy and can affect in retinal involvement in the pathological process and development of proliferative retinopathy [14].
Chartens and Lightman have reported that IFN-gamma is produced by lymphocytes infiltrating the retina and vascular membrane, which is associated with a destructive process and indicates disease progression [15]. IFN-gamma enhances the cytotoxic effect of TNF-alpha on pigment epithelial cells.
A two-fold higher IL-10 concentration was revealed in the subretinal fluid of patients with 3-4 week detachment compared to 5-8 week and over 2 month detachment, the elevated level being preceded by the maximum concentrations of such pro-inflammatory cytokines as IL-8 and TNF-alpha.

In experimental chorioretinitis using the animal model, IL-10 simultaneously administered with LPS to the vitreous showed a protective effect. The inhibition of the inflammatory reaction was considerably reduced when the cytokine administration was delayed compared with LPS. IL-10 when administered with the recombinant IL-1 did not prevent vascular membrane inflammation [16]. A pro-inflammatory action of this cytokine has been recently demonstrated in vivo and in vitro. It seems that its final biological effect is dependent, among other things, on the cytokine signal sequence [17, 18].
The IL-13, which inhibits the production of pro-inflammatory cytokines such as TNF-alpha, IL-1 alpha and beta, IL-6 and IL-8, was present in SRF in concentrations a few times higher than IL-10, its level showing a negative correlation with detachment duration [19].
In conclusion, it should be noted that the presence of cytokines in the subretinal fluid and concentration dynamics in time indicate the possibility of their local production by SRF cells and retinal pigment epithelial cells, and suggest their significant involvement in the pathogenesis of rhegmatogenous retinal detachment, especially that complicated by PVR.


References


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Correspondence: Małgorzata Mrugacz, MD, Department of Paediatric Ophthalmology, Medical Academy of Białystok, Waszyngton 17, str.,
15-274 Białystok, Poland. Phone number: +48 85 745 08 62, e-mail: mrugacz1@poczta.onet.pl