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Keywords:

  • cytokines;
  • epithelial growth factor;
  • exudative age-related macular degeneration;
  • intercellular adhesion molecule-1;
  • monocyte chemoattractant protein-1

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Financial disclosures
  8. References

Purpose:  To measure the concentration of cytokines in the aqueous humour of eyes with exudative age-related macular degeneration (AMD).

Methods:  The clinical interventional study included a study group of 18 patients with exudative AMD and a control group of 20 patients undergoing routine cataract surgery. Age did not vary significantly (p = 0.36) between study group (80.8 ± 6.4 years) and control group (77.0 ± 9.9 years), nor did gender (p = 0.75). During the interventions, aqueous humour samples were obtained, in which the concentration of cytokines was measured using a solid-phase chemiluminescence immunoassay. Macular thickness was measured by optical coherence tomography (OCT).

Results:  In the study group as compared to the control group, significantly higher concentrations were measured for epithelial growth factor (EGF) (p = 0.017), human growth factor (HGF) (p = 0.048), intercellular adhesion molecule-1 (ICAM1) (p = 0.028), interleukin 12p40 (IL12p40) (p = 0.009), interleukin 1a2 (IL1a2) (p = 0.01), interleukin 3 (IL3) (p = 0.02), interleukin 6 (IL6) (p = 0.006), interleukin 8 (IL8) (p = 0.02), monocyte chemoattractant protein-1 (MCP-1) (p = 0.048), monokine induced by interferon gamma (MIG) (p = 0.016), matrix metalloproteinase 9 (MMP9) (p = 0.004) and plasminogen activator inhibitor 1 (PAI1) (p = 0.006). Macular thickness was significantly associated with the concentrations of EGF (p = 0.001), HGF (p = 0.02), ICAM1 (p = 0.001), interleukin 12p40 (p = 0.006), IL 1a2 (p = 0.002), MIG (p = 0.001), MMP9 (p < 0.001) and PAI1 (p = 0.01). Interleukin 6 and MCP-1 showed significant associations with the height of retinal pigment epithelium detachment.

Conclusions:  Numerous cytokines are associated with the presence and the amount of exudative AMD.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Financial disclosures
  8. References

Intraocular neovascular or oedematous diseases such as diabetic retinopathy and exudative age-related macular degeneration (AMD) belong to the leading causes of severe, often irreversible visual impairment in the elderly population (De Jong 2006; Wong & Hyman 2008). After the landmark studies by Aiello and colleagues on the association of retinal neovascularization with the vascular endothelium growth factor (VEGF) in diabetic retinopathy and retinal vein occlusions (Aiello et al. 1994, 1995), consequent investigations showed the associations of VEGF with other neovascular diseases such as exudative AMD and retinopathy of prematurity (Kvanta et al. 1996; Matsuoka et al. 2004; Nonobe et al. 2009; Sato et al. 2009). Other studies revealed that besides VEGF, also other cytokines were found in elevated concentrations in the aqueous humour, vitreous and retina of eyes with neovascular disorders. The list of these cytokines included erythropoietin (Katsura et al. 2005; Watanabe et al. 2005; Jonas & Neumaier 2007a) basic fibroblast growth factor (Jonas & Neumaier 2007b), pigment epithelial-derived factor (Mori et al. 2002; Cox et al. 2003; Chan et al. 2008), monocyte chemoattractant protein-1 (MCP-1) (Takeda et al. 2009; Jonas et al. 2010), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (Shen et al. 1998; Jonas et al. 2010), protein kinase C (Kociok et al. 1998; Saishin et al. 2003; Yokota et al. 2003), platelet-derived growth factor (Robbins et al. 1994; Vinores et al. 1995) and others (Kliffen et al. 1997; Funk et al. 2009a,b). Knowledge about mediators in the intraocular neovascular and oedematous process is of importance for the development of new treatment strategies, as has been shown by the clinical application of ranibizumab and bevacizumab as VEGF inhibitors in the therapy of exudative AMD as outstanding example (Rosenfeld et al. 2006). We therefore performed this study to search for additional substances that may be present in abnormal concentrations in the aqueous humour of patients with exudative AMD.

Methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Financial disclosures
  8. References

The clinical interventional comparative study included a study group consisting of patients with exudative AMD who were treated by an intravitreal injection of bevacizumab and/or triamcinolone (Jonas et al. 2005), and a control group of patients with age-related cataract who underwent routine cataract surgery. All patients were treated at the same institution. Inclusion criterion for both groups was the absence of any retinal or optic nerve disease except of exudative AMD in the study group. Additionally, the volume of the collected aqueous humour had to be at least 100 μl. A previous photodynamic therapy or laser photocoagulation as therapy of the subfoveal neovascular membrane was an exclusion criterion, while previous intravitreal drug applications (bevacizumab or triamcinolone acetonide) were allowed if they had taken place at least 6 months prior to the inclusion into the study. Intraocular pressure had to be within the normal range of 10–21 mmHg.

All patients underwent an ophthalmologic examination including refractometry, applanation tonometry and slit lamp-assisted biomicroscopy of the anterior segment and posterior segment of the eye. The diagnosis of exudative AMD was substantiated by ophthalmoscopy, fluorescein angiogram and optical coherence tomography (OCT III Stratus, Carl-Zeiss-Meditec, Jena, Germany). Using the OCT imaging, we differentiated between, and measured separately, the thickness of the foveal retina (‘retinal Thickness Only’), the distance between the inner limiting membrane and the retinal pigment epithelium (‘macular thickness plus subretinal fluid without retinal pigment epithelium elevation’), and the height of retinal pigment epithelium detachment. Reason for this differentiation was that potentially different cytokines or pathomechanisms may be responsible for the morphologically different types of macular oedema.

In the study group, the aqueous humour was collected after disinfection of the periorbital skin and conjunctiva, sterile draping of the patient and insertion of a lid speculum. A paracentesis was performed in the temporal limbal region, and aqueous humour was sampled using a blunt canula and a tuberculin syringe, before the intravitreal injection was transconjuntivally performed in the temporal inferior quadrant. The release of aqueous humour was necessary because the volume of the drug (triamcinolone acetonide) to be injected (0.2 ml) was too large to fit into a globe without prior release of fluid. These injections were performed before intravitreal bevacizumab was used for the therapy of exudative AMD. In the control group, the periorbital skin was disinfected, the patient was draped, a lid speculum was inserted, and the aqueous humour was collected through a temporal paracentesis, before routine cataract surgery was continued. For all patients, the aqueous humour samples were deeply frozen in liquid nitrogen within 10 min after collection. In the study group, the paracentesis and the release of aqueous humour was necessary, because these patients underwent an injection of triamcinolone or a combined injection of bevacizumab and triamcinolone with a total injected volume of about 0.20–0.25 ml (Jonas et al. 2005). For such a volume to be injected intravitreally, the eye had to become hypotonous or to the injection to avoid a marked injection-related increase in intraocular pressure. In the control group, the paracentesis was routinely performed to create a temporal access to the anterior chamber for bimanual manoeuvring of the lens nucleus and cortex during surgery. The technique has been described previously (Jonas et al. 2003, 2005).

Samples were analysed using the Luminex xMAP suspension array technology (Luminex Co., Austin, Texas, USA) (Funk et al. 2010). A custom-made kit (Progen Co., Heidelberg, Germany) was used for the detection of TGFα, TGF-ß, EGF, FGFß, HGF, IFNα, IFNβ, IFNγ, IL1a2, IL1b, IL2, IL3, IL4, IL5, IL6, IL8, IL10, IL12p40, IL12p70, IP10, ICAM1, MCP-1, MCP-3, MIF, MIG, MMP1, MMP3, MMP9, PAI1, PlGF, PDGF-BB, SDF1, TNF, TRAIL, VCAM and VEGF (Table 1). The reasons to choose these cytokines for measurement were results of previous investigations (Aiello et al. 1994, 1995; Chan et al. 2008; Cox et al. 2003; Funk et al. 2009a; b; Jonas & Neumaier 2007a; b; Jonas et al. 2010; Katsura et al. 2005; Kliffen et al. 1997; Kociok et al. 1998; Mori et al. 2002; Robbins et al. 1994; Saishin et al. 2003; Shen et al. 1998; Takeda et al. 2009; Vinores et al. 1995; Watanabe et al. 2005; Yokota et al. 2003) and reflections that these cytokines may be the mostly like substances to show differences between the study group and the control group. Aqueous humour samples (50 μl) were used undiluted and incubated overnight. The kit was run according to the manufacturer’s instructions. Standard curves for each cytokine (in duplicate) were generated using the reference cytokine concentrations supplied in this kit. All incubation steps were performed at room temperature and in the dark to protect the beads from light. Samples were read on the Luminex xMAP system. To avoid between-run imprecision, all samples from the same individual before and after the interventions were measured in the same run. Control samples were included in all runs. The detection limit for any analyte was 0.61 pg/ml with a dynamic range up to 10 000 pg/ml (according to the manufacturers).

Table 1.   Aqueous humour concentrations (pg/ml) (mean; median; range) of cytokines in patients with exudative age-related macular degeneration (AMD) and subjects undergoing routine cataract surgery.
CytokineExudative AMD study groupCataract control groupp-value
  1. n = number of samples with values above level of detectability.

  2. p-value: statistical significance of the difference between both groups.

Epidermal growth factor6.62 (5.70; 0.00, 23.4)4.22 (4.38; 0.00, 7.90)0.017
Basic fibroblast growth factor (bFGF)0.69 (0.00; 0.00, 12.4) (n = 1)00.78
Human growth factor257 (126; 34, 1594)125 (89; 47, 687)0.048
Intercellular adhesion molecule-1 (ICAM1)646 (400; 40, 3090)335 (235; 83, 1214)0.028
Interferon alpha (IFNa)1.05 (0.00; 0.00, 10.1)00.39
Interferon beta (IFNb)001.0
Interferon gamma (IFNg)001.0
Interleukin 12p40 (IL12p40)3.00 (2.35; 0.00, 8.08)1.59 (1.23; 0.00, 5.30)0.009
Interleukin 12p70 (IL12p70)0.61 (0.00; 0.00, 3.28)0.54(0.00; 0.00, 3.28)0.43
Interleukin 1a2 (IL1a2)16.0 (6.54; 0.00, 68.4)4.32 (2.33; 0.00, 29.3)0.01
Interleukin 1b (IL1b)0.44 (0.00; 0.00, 7.94) (n = 1)00.78
Interleukin 2 (IL2)001.0
Interleukin 3 (IL3)1.93 (1.36; 0.00, 6.42)0.38 (0.00; 0.00, 4.06)0.02
Interleukin 4 (IL4)0.08 (0.00; 0.00, 1.42) (n = 2)00.78
Interleukin 5 (IL5)0.29 (0.00; 0.00, 2.10)0.05 (0.00; 0.00, 1.02)0.52
Interleukin 6 (IL6)154 (10.1; 1.02, 1309)6.42 (2.65; 0.00, 69.1)0.006
Interleukin 8 (IL8)3.34 (1.56; 0.00, 19.6)0.43 (0.00; 0.00, 1.98)0.02
Interleukin 10 (IL10)001.0
Interferon-gamma-induced protein (IP10)70.2 (31.3; 5.76, 270)33.4 (25.1; 7.68, 128)0.28
Monocyte chemoattractant protein-1 (MCP-1)267 (233; 51, 825)177 (142; 58, 396)0.048
Monocyte chemoattractant protein-3 (MCP-3)4.46 (0.00; 0.00, 21.9)1.10 (0.00; 0.00, 21.9)0.16
Macrophage migration inhibitory factor5584 (3883;467, 18 082)4931 (3059; 210, 17 500)0.68
Monokine induced by interferon gamma62.6 (48.9; 15.1, 188)36.7 (37.8; 11.8, 60.9)0.016
Matrix metalloproteinase 1 (MMP1)1.75 (0.00; 0.00, 10.1)0.96 (0.00; 0.00, 17.5)0.33
Matrix metalloproteinase 9 (MMP9)54.8 (35.1; 0.00, 288)17.5 (6.80; 0.00, 112)0.004
Plasminogen activator inhibitor 1 (PAI1)281 (116; 13, 1472)137 (65.4; 18.7, 1193)0.006
Platelet-derived growth factor (PDGF)0.93 (0.00; 0.00, 16.9) (n = 2)00.78
Placenta growth factor3.44 (1.52; 0.00, 10.9)1.65 (0.00; 0.00, 10.0)0.16
Stromal cell-derived factor 1 (SDF1)2.04 (0.00; 0.00, 9.15)0.95 (0.00; 0.00, 5.79)0.44
Transforming growth factor alpha (TGFa)0.16 (0.00; 0.00, 1.24)00.39
Transforming growth factor beta (TGFb)23.8 (18.1; 5.36, 74.9)18.3 (16.0; 6.1, 63.9)0.25
Tumour necrosis factor alpha-related apoptosis-inducing ligand2.99 (1.40; 0.00,20.1)1.25 (0.44; 0.00, 3.96)0.36
Vascular cell adhesion molecule1874 (971;163, 15 988)765 (736; 167, 1645)0.15
Vascular endothelial growth factor31.4 (21.8; 0.00, 87.4)41.4 (40.5; 0.00, 102)0.10

Statistical analysis was performed using a commercially available statistical software package (spss for Windows, version 19.0, SPSS, Chicago, IL, USA). The two study groups were compared with each other using the nonparametric Mann–Whitney test.

Results

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Financial disclosures
  8. References

The study group included 18 patients (nine women) with exudative AMD and the control group consisted of 20 patients (eight women) with cataract. Age did not vary significantly (p = 0.36) between the study group (80.8 ± 6.4 years; median: 81.9 years; range: 64.8–87.8 years) and the control group (77.0 ± 9.9 years; median: 77.3 years; range: 62.1–92.1 years), nor did gender (p = 0.75).

In the study group as compared to the control group, significantly higher concentrations were measured for epithelial growth factor (EGF) (p = 0.017), human growth factor (HGF) (p = 0.048), intercellular adhesion molecule-1 (ICAM1) (p = 0.028), interleukin IL12p40 (IL12p40) (p = 0.009), interleukin 1a2 (IL1a2) (p = 0.01), interleukin 3 (IL3) (p = 0.02), interleukin 6 (IL6) (p = 0.006), interleukin 8 (IL8) (p = 0.02), monocyte chemoattractant protein-1 (MCP-1) (p = 0.048), monokine induced by interferon gamma (MIG) (p = 0.016), matrix metalloproteinase 9 (MMP9) (p = 0.004) and plasminogen activator inhibitor 1 (PAI1) (p = 0.006) (Table 1).

Both groups did not vary significantly in the concentrations of basic fibroblast growth factor beta (FGFß), interferon alpha (IFNa), interferon beta (IFNb) and interferon gamma (IFNg), interleukin IL12p70), interleukin 1b, 2, 4, 5 and 10, interferongamma-induced protein (IP10), monocyte chemoattractant protein-3 (MCP-3), macrophage migration inhibitory factor (MIF), matrix metalloproteinase 1 (MMP1), platelet-derived growth factor (PDGF-BB), placenta growth factor (PLGF), stromal cell-derived factor 1 (SDF1), tissue growth factor alpha (TGFa) and beta (TGFb), TNF(tumour necrosis factor alpha)-related apoptosis-inducing ligand (TRAIL), vascular cell adhesion molecule (VCAM) and vascular endothelial growth factor (VEGF) (Table 1).

Optical coherence tomograms were available for all patients included into the study. The mean retinal macular thickness (defined as distance between inner limiting membrane and outer surface of the retinal photoreceptors) was 476 ± 181 μm (median: 433 μm; range: 216–865 μm); the mean total macular thickness [defined as distance between the inner limiting membrane and the surface of the retinal pigment epithelium (RPE)] (i.e. including the height of subretinal fluid) was 541 ± 282 μm (median: 433 μm; range: 216–1298 μm); and the mean height of a retinal pigment epithelium detachment (defined as distance between the actual position of the retinal pigment epithelium and the normal level of the retinal pigment epithelium) was 303 ± 466 μm (median: 87 μm; range: 0–1730 μm). The retinal macula thickness for the subjects of the control group was set at 212 μm (Chan et al. 2006).

Correlating the aqueous humour concentrations of the cytokines with the thickness of the macula defined as distance between the retinal inner limiting membrane and the retinal pigment epithelium layer, significant associations were found for the concentrations of the epidermal growth factor (EGF), HGF, intercellular adhesion molecule-1 (ICAM1), interleukin 12p40, interleukin 1a2, MIG, matrix metalloproteinase 9 (MMP9) and plasminogen activator inhibitor 1 (PAI1) (Table 2). Interleukin 6 and monocyte chemoattractant protein-1 (MCP-1) (Fig. 1) showed significant associations with the height of retinal pigment epithelium detachment (Table 2) (Figs 2–4).

Table 2.   Correlations between the thickness of the macula (defined as distance between inner limiting membrane and outer surface of the retinal photoreceptors), or the total macular thickness [defined as distance between the inner limiting membrane and the surface of the retinal pigment epithelium (RPE)] (i.e. including the height of subretinal fluid), or the height of a retinal pigment epithelium detachment (defined as distance between the actual position of the retinal pigment epithelium and the normal level of the retinal pigment epithelium) and the concentrations of cytokines in the aqueous humour in a study group of patients with exudative age-related macular degeneration and a control group of patients undergoing routine cataract surgery.
CytokineMacular thickness (only retinal tissue)Macular thickness plus subretinal fluid, without RPE elevationRPE height
Correlation coefficient r (r2)p-valueCorrelation coefficient r (r2)p-valueCorrelation coefficient r (r2)p-value
Epidermal growth factor0.54 (0.29)0.0010.52 (0.27)<0.0010.77
Human growth factor0.39 (0.15)0.020.080.95
Intercellular adhesion molecule-10.51 (0.26)0.0010.48 (0.23)0.0020.93
Interleukin 12p40 (IL12p40)0.44 (0.19)0.0060.55 (0.30)<0.0010.44
Interleukin 1a2 (IL1a2)0.48 (0.23)0.0020.37 (0.14)0.020.46 (0.21)0.004
Interleukin 3 (IL3)0.430.370.61
Interleukin 6 (IL6)0.320.600.64 (0.41)<0.001
Interleukin 8 (IL8)0.110.120.56
Monocyte Chemoattractant Protein-1 (MCP-1)0.070.130.46 (0.21)0.002
Monokine induced by interferon gamma0.53 (0.28)0.0010.61 (0.37)<0.0010.09
Matrix metalloproteinase 9 (MMP9)0.070.68 (0.46)<0.0010.95
Plasminogen activator inhibitor 1 (PAI1)0.40 (0.16)0.010.46 (0.21)0.0040.67
image

Figure 1.  Scatterplot showing the association between the height of the retinal pigment epithelium detachment and the aqueous humour concentration of monocyte chemoattractant protein-1 (MCP-1) [equation of the regression line: MCP-1 Concentration (pg/ml) = 0.20 × macular thickness (μm) + 198; correlation coefficient r = 0.46 (r2 = 0.21); p = 0.002].

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image

Figure 2.  Scatterplot showing the association between the thickness of the macula defined as distance between the retinal inner limiting membrane and the retinal pigment epithelium layer and the aqueous humour concentration of epithelial growth factor (EGF) [equation of the regression line: EGF Concentration (pg/ml) = 0.008 × macular thickness (μm) + 2.60; correlation coefficient r = 0.52 (r2 = 0.27); p < 0.001].

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image

Figure 3.  Scatterplot showing the association between the thickness of the macula defined as distance between the retinal inner limiting membrane and the retinal pigment epithelium layer and the aqueous humour concentration of intercellular adhesion molecule-1 (ICAM1) [equation of the regression line: ICAM-1 Concentration (pg/ml) = 0.99 × macular thickness (μm) + 116.2; correlation coefficient r = 0.48 (r2 = 0.23); p = 0.001].

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image

Figure 4.  Scatterplot showing the association between the thickness of the macula defined as distance between the retinal inner limiting membrane and the retinal pigment epithelium layer and the aqueous humour concentration of monokine induced by interferon gamma [equation of the regression line: MIG Concentration (pg/ml) = 0.096 × macular thickness (μm) + 17.4; correlation coefficient r = 0.61 (r2 = 0.37); p < 0.001].

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Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Financial disclosures
  8. References

The results of our study suggest that eyes with exudative AMD show an abnormally high level of a whole array of cytokines in their aqueous humour. The list of these cytokines includes EGF, HGF, intercellular adhesion molecule-1 (ICAM1), interleukin 12p40, interleukin 1a2, interleukin 3, interleukin 6, interleukin 8, monocyte chemoattractant protein-1 (MCP-1), MIG, matrix metalloproteinase 9 (MMP9) and plasminogen activator inhibitor 1 (PAI1). Interestingly, the concentration of vascular endothelial growth factor (VEGF) did not vary significantly (p = 0.10) between the study group and control group.

Some of these findings confirm previous investigations, and some of the results represent new findings. To address some of the cytokines examined, in recent studies as in our investigations, eyes with exudative AMD showed elevated levels of monocyte chemoattractant protein-1 (MCP-1) (Takeda et al. 2009; Jonas et al. 2010). Previous studies revealed that inflammation is critically involved in the formation of subfoveal choroidal neovascular membranes and may thus contribute to the pathogenesis of AMD (Anderson et al. 2002; Donoso et al. 2006). The recruitment of monocytes is an early step in the initiation of the inflammatory and angiogenic processes, and MCP-1 plays an important role in regulation of the migration and infiltration of monocytes and macrophages (Deshmane et al. 2009). MCP-1 has been known to be constitutively released from the retinal pigment epithelium and is enhanced by pro-inflammatory molecules (Crane et al. 2000; Forrester 2003). Recently, it was put forward by Austin and colleagues that the enhanced release of MCP-1 could attract resident ocular macrophages (microglia) and could contribute to the digestion of the retinal pigment epithelium and Bruch’s membrane as its basal membrane (Austin et al. 2009). Correspondingly, histologic examinations of surgically removed subfoveal neovascular membranes from patients with exudative AMD showed macrophages located in regions with an atrophy of the retinal pigment epithelium, Bruch’s membrane breakdown and choroidal neovascularization (Penfold et al. 1985; Killingsworth et al. 1990; Hutchinson et al. 1993; Dastgheib & Green 1994; Patel & Chan 2008). These data suggest that MCP-1 essentially contributes to the formation of subfoveal choroidal neovascular membranes. Additionally, MCP-1 has been known to facilitate angiogenesis (Voskuil et al. 2003; Hong et al. 2005). Yamada and coworkers demonstrated that VEGF increases MCP-1 mRNA levels in cultured endothelial cells and that MCP-1 participates in VEGF-induced angiogenesis and vascular leakage, which may be helpful to explain our result that aqueous concentration of MCP-1 positively correlated with maximum retinal thickness of the macula in the patients with exudative AMD (Yamada et al. 2003).

Besides MCP-1, another molecule of special interest is intercellular adhesion molecule-1 (ICAM1). Our study revealed that the eyes with exudative AMD showed significantly higher levels of ICAM1 (Tables 1 and 2). This molecule belongs to the immunoglobulin superfamily (IgSF) and is a key indicator of vascular endothelial cell activation. Adhesion molecule expression, including ICAM-1, has been found in association with inflammatory cells in extracted subretinal neovascular membranes lesions (Heidenkummer & Kampik 1995). Moreover, ICAM-1 labelling of the choriocapillaris was typically more intense in the macula than in the peripheral choroid in human donor eyes. It implies that the macula may be subject to an increased leucocyte trafficking (Mullins et al. 2006). In agreement with these previous findings, our results showed that the intraocular concentrations of ICAM-1 were significantly higher in the patients with exudative AMD than in the patients of the control group.

On the basis of the landmark studies by Aiello and colleagues (Aiello et al. 1994), vascular endothelial growth factor (VEGF) is a cytokine of special interest. Our finding that the aqueous concentration of VEGF did not vary significantly between the patients with exudative AMD and the cataract patients is in agreement with a study by Chan and colleagues (Chan et al. 2008), in which the aqueous VEGF concentration did not differ significantly between a study group with untreated choroidal neovascular neovascularization, study group with recurrent choroidal neovascular neovascularization and a control group consisting of patients with cataract (p = 0.31 and p = 0.21, respectively). The result of our study also agrees with the finding of our previous study on different patients in that the aqueous concentration of VEGF was not significantly different between a group of patients with exudative AMD and a control group (Jonas & Neumaier 2007b). With respect to the concentration of VEGF, the results of our study also agree with a recent investigation by Roh and colleagues who collected aqueous humour samples from 36 eyes with AMD and 10 controls during cataract surgery (Roh et al. 2009). Of 36 patients with AMD, five eyes were naïve to an intravitreal bevacizumab injection, 14 eyes had a recurrent choroidal neovascular membrane after bevacizumab treatment, and 17 eyes had a regressed choroidal neovascular membrane after bevacizumab treatment. Vascular endothelium growth factor in both naive and recurrent CNV groups was significantly higher compared with regressed CNV group (p = 0.025 and p = 0.004, respectively) but was not statistically different from the control group (p = 0.31 and p = 0.21, respectively). The aqueous humour level of interleukin (IL)-2 was significantly lower in the recurrent choroidal neovascular membrane group (p = 0.036 and p = 0.019) compared with the control group. In the active choroidal neovascular membrane patients (recurrent and naïve choroidal neovascular membrane groups), the aqueous humour levels of IL-6 and IL-8 significantly correlated with the size of the choroidal neovascular membrane. Besides the differences between the control group and the treated groups, the authors interestingly did not find significant differences in the cytokine levels between the control group and the naïve choroidal neovascular membrane group.

Findings of our study which have not been widely reported in previous investigations were the association between exudative AMD and elevated intraocular levels of EGF, interleukin 1a2 (IL 1a2), interleukin 3 (IL3), interleukin 6 (IL6), interleukin 8 (IL8), interleukin IL12p40 (IL12p40), monocyte chemoattractant protein-3 (MCP-3), matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 9 (MMP9), plasminogen activator inhibitor 1 (PAI1) and transforming growth factor beta (TGF-ß).

Interestingly, the cytokines differed in their associations with an intraretinal and subretinal retinal oedema versus a detachment of the retinal pigment epithelium (Table 2). While the amount of retinal oedema was associated with elevated concentrations of ICAM-1, IL12p40, IL1a2, MIG, MCP-1, PAI1, EGF and HGF, the height of a detachment of the retinal pigment epithelium was associated with the levels of MCP-1, IL6 and IL1a2 (Table 2). If these findings are confirmed by future studies, they may suggest different pathomechanisms for retinal oedema versus a detachment of the retinal pigment epithelium and may then potentially offer different treatment strategies.

In the interpretation of the findings, one may be cautious to conclude that elevated concentrations of cytokines in the eyes with exudative AMD were causally related with exudative AMD and may thus be therapeutic targets. The elevated levels of the cytokines may be due to several reasons, such as a retinal leakage because of an insufficient blood retina barrier that is also responsible for the macular oedema in eyes with exudative AMD; and last but not least a local production or hyperproduction of the cytokines in the diseased retina and choroid. In that scenario, development of procedures neutralizing these cytokines may be therapeutically helpful, as shown by bevacizumab and ranibizumab to neutralize vascular endothelial growth factor. The findings of our study may serve to further examine the reasons for the associations of specific cytokines with exudative AMD, for example by performing immunohistochemical studies on the retina of animals with induced choroidal neovascularization, on post-mortem human eyes with exudative AMD or on subfoveal membranes removed during surgery for exudative AMD (Roh et al. 2009; Takeda et al. 2009). One of the reasons why anti-VEGF drugs such as ranibizumab and bevacizumab have clinically a markedly more pronounced anti-angiogenic effect in proliferative diabetic retinopathy than in exudative AMD may be that aqueous humour concentrations of VEGF were markedly more elevated in eyes with proliferative diabetic retinopathy than in eyes with exudative AMD as compared to control eyes. It may suggest that other cytokines besides VEGF may play a role in the pathogenesis of exudative AMD and may potentially be therapeutic targets.

Limitations of our study should be mentioned. First, our study only examined whether cytokines showed an increased concentration in the aqueous humour, and whether these measurements were correlated with morphometric measurement of the macula. Because macular diseases are associated with a breakdown in the blood retina barrier, it has remained unclear whether the increased concentrations of the cytokines in the aqueous humour were because of a leakage effect through the damage vascular walls, or whether the cytokines were actively produced in the eye and were thus causally related with the development of the exudative AMD. Second, the number of patients enrolled into our study was relatively low. Despite it, the results were statistically significant, so that the relatively small number of patients may serve to strengthen the results and conclusions of the study. Third, the concentration of the cytokines was determined from aqueous samples and not from vitreal samples that usually show higher concentrations and may better reflect the situation of the retina and choroid (Kamppeter et al. 2008). Obtaining vitreous samples in the patients was however not possible because it would have necessitated an intravitreal intervention. Fourth, the study group and control group were too small for a meaningful multivariate analysis, so that it has remained inconclusive whether inter-dependencies between the various cytokines influenced the results. Fifth, because of the limited volume of the aqueous humour samples, we could not determine all cytokines that may be associated with exudative AMD. Molecules not examined but potentially associated with the disease include 8-hydroxy-2′-deoxyguanosine (Lau et al. 2010), pigment epithelium-derived growth factor PEGF (King & Suzuma 2000) and others.

In conclusion, the aqueous level of various cytokines including growth factors, interleukins, monocyte chemoattractant protein-1, adhesion molecules and matrix metalloproteinases was elevated in eyes with exudative AMD. These data may be of interest to find cytokines associated with exudative AMD in addition to those already known to be related with the disease. It may help to develop new treatment strategies neutralizing cytokines found in abnormally high concentrations in eyes with exudative AMD.

Financial disclosures

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Financial disclosures
  8. References

Jost B. Jonas – Consultant for Allergan Inc.; Merck & Co., Inc.; Pfizer Inc.; Bayer-Schering Co; CellMed AG, Alzenau, Germany; Morphosys, Munich, Germany; SOOFT SpA Montegiorgio, Italy; Patent holder with CellMed AG, Alzenau, Germany. All other authors: None

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Financial disclosures
  8. References
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