Cytokine concentration in aqueous humour of ... - Wiley Online Library

Acta Ophthalmologica 2012

Cytokine concentration in aqueous humour of eyes with exudative age-related macular degeneration Jost B. Jonas,1 Yong Tao,2 Michael Neumaier3 and Peter Findeisen3 1

Department of Ophthalmology, Medical Faculty Mannheim of the Ruprecht-KarlsUniversity Heidelberg, Germany 2 Department of Ophthalmology, People’s Hospital, Peking University, Beijing, China 3 Institute for Clinical Chemistry, Medical Faculty Mannheim of the RuprechtKarls-University Heidelberg, Germany

ABSTRACT. 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 solidphase 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. Key words: cytokines – epithelial growth factor – exudative age-related macular degeneration – intercellular adhesion molecule-1 – monocyte chemoattractant protein-1

Acta Ophthalmol. 2012: 90: e381–e388 ª 2012 The Authors Acta Ophthalmologica ª 2012 Acta Ophthalmologica Scandinavica Foundation

doi: 10.1111/j.1755-3768.2012.02414.x

Introduction 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.

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2010), intercellular adhesion molecule1 (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 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 agerelated 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 ll. 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

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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 injectionrelated increase in intraocular pressure. In the control group, the paracentesis was routinely performed to create a temporal access to the ante-

rior 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 TGFa, TGF-ß, EGF, FGFß, HGF, IFNa, IFNb, IFNc, 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 ll) 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). 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.


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.

Cytokine Epidermal growth factor Basic fibroblast growth factor (bFGF) Human growth factor Intercellular adhesion molecule-1 (ICAM1) Interferon alpha (IFNa) Interferon beta (IFNb) Interferon gamma (IFNg) Interleukin 12p40 (IL12p40) Interleukin 12p70 (IL12p70) Interleukin 1a2 (IL1a2) Interleukin 1b (IL1b) Interleukin 2 (IL2) Interleukin 3 (IL3) Interleukin 4 (IL4) Interleukin 5 (IL5) Interleukin 6 (IL6) Interleukin 8 (IL8) Interleukin 10 (IL10) Interferon-gamma-induced protein (IP10) Monocyte chemoattractant protein-1 (MCP-1) Monocyte chemoattractant protein-3 (MCP-3) Macrophage migration inhibitory factor Monokine induced by interferon gamma Matrix metalloproteinase 1 (MMP1) Matrix metalloproteinase 9 (MMP9) Plasminogen activator inhibitor 1 (PAI1) Platelet-derived growth factor (PDGF) Placenta growth factor Stromal cell-derived factor 1 (SDF1) Transforming growth factor alpha (TGFa) Transforming growth factor beta (TGFb) Tumour necrosis factor alpha-related apoptosis-inducing ligand Vascular cell adhesion molecule Vascular endothelial growth factor

Exudative AMD study group

Cataract control group

p-value

6.62 (5.70; 0.00, 23.4) 0.69 (0.00; 0.00, 12.4) (n = 1) 257 (126; 34, 1594) 646 (400; 40, 3090)

4.22 (4.38; 0.00, 7.90) 0

0.017 0.78

125 (89; 47, 687) 335 (235; 83, 1214)

0.048 0.028

1.05 (0.00; 0.00, 10.1) 0 0 3.00 (2.35; 0.00, 8.08) 0.61 (0.00; 0.00, 3.28) 16.0 (6.54; 0.00, 68.4) 0.44 (0.00; 0.00, 7.94) (n = 1) 0 1.93 (1.36; 0.00, 6.42) 0.08 (0.00; 0.00, 1.42) (n = 2) 0.29 (0.00; 0.00, 2.10) 154 (10.1; 1.02, 1309) 3.34 (1.56; 0.00, 19.6) 0 70.2 (31.3; 5.76, 270)

0 0 0 1.59 (1.23; 0.00, 5.30) 0.54(0.00; 0.00, 3.28) 4.32 (2.33; 0.00, 29.3) 0

0.39 1.0 1.0 0.009 0.43 0.01 0.78

0 0.38 (0.00; 0.00, 4.06) 0

1.0 0.02 0.78

0.05 6.42 0.43 0 33.4

0.52 0.006 0.02 1.0 0.28

(0.00; 0.00, 1.02) (2.65; 0.00, 69.1) (0.00; 0.00, 1.98) (25.1; 7.68, 128)

267 (233; 51, 825)

177 (142; 58, 396)

0.048

4.46 (0.00; 0.00, 21.9)

1.10 (0.00; 0.00, 21.9)

0.16

5584 (3883;467, 18 082)

4931 (3059; 210, 17 500)

0.68

62.6 (48.9; 15.1, 188)

36.7 (37.8; 11.8, 60.9)

0.016

1.75 (0.00; 0.00, 10.1)

0.96 (0.00; 0.00, 17.5)

0.33

54.8 (35.1; 0.00, 288)

17.5 (6.80; 0.00, 112)

0.004

281 (116; 13, 1472)

137 (65.4; 18.7, 1193)

0.006

0.93 (0.00; 0.00, 16.9) (n = 2) 3.44 (1.52; 0.00, 10.9) 2.04 (0.00; 0.00, 9.15)

0

0.78

1.65 (0.00; 0.00, 10.0) 0.95 (0.00; 0.00, 5.79)

0.16 0.44

0.16 (0.00; 0.00, 1.24)

0

0.39

23.8 (18.1; 5.36, 74.9)

18.3 (16.0; 6.1, 63.9)

0.25

2.99 (1.40; 0.00,20.1)

1.25 (0.44; 0.00, 3.96)

0.36

1874 (971;163, 15 988)

765 (736; 167, 1645)

0.15

31.4 (21.8; 0.00, 87.4)

41.4 (40.5; 0.00, 102)

0.10

n = number of samples with values above level of detectability. p-value: statistical significance of the difference between both groups.

Results 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 con-

trol 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), plateletderived 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 lm (median: 433 lm; range: 216–865 lm); 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 lm (median: 433 lm; range: 216–1298 lm); and the mean height of a retinal pigment epithelium detachment (defined as distance between the actual position of the

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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.

Cytokine Epidermal growth factor Human growth factor Intercellular adhesion molecule-1 Interleukin 12p40 (IL12p40) Interleukin 1a2 (IL1a2) Interleukin 3 (IL3) Interleukin 6 (IL6) Interleukin 8 (IL8) Monocyte Chemoattractant Protein-1 (MCP-1) Monokine induced by interferon gamma Matrix metalloproteinase 9 (MMP9) Plasminogen activator inhibitor 1 (PAI1)

Macular thickness (only retinal tissue)

Macular thickness plus subretinal fluid, without RPE elevation

RPE height

Correlation coefficient r (r2)

p-value


p-value


p-value

0.54 0.39 0.51 0.44 0.48 – – – –

(0.29) (0.15) (0.26) (0.19) (0.23)

0.001 0.02 0.001 0.006 0.002 0.43 0.32 0.11 0.07

0.52 – 0.48 0.55 0.37 – – – –