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Immunology and Microbiology  |   October 2012
Cytokines and Chemokines in the Vitreous Fluid of Eyes with Uveal Melanoma
Author Affiliations & Notes
  • Nisha Nagarkatti-Gude
    From the Departments of Ophthalmology and
  • Inge H. G. Bronkhorst
    From the Departments of Ophthalmology and
  • Sjoerd G. van Duinen
    Pathology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
  • Gregorius P. M. Luyten
    From the Departments of Ophthalmology and
  • Martine J. Jager
    From the Departments of Ophthalmology and
  • Corresponding author: Martine J. Jager, Department of Ophthalmology, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands; m.j.jager@lumc.nl
Investigative Ophthalmology & Visual Science October 2012, Vol.53, 6748-6755. doi:10.1167/iovs.12-10123
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      Nisha Nagarkatti-Gude, Inge H. G. Bronkhorst, Sjoerd G. van Duinen, Gregorius P. M. Luyten, Martine J. Jager; Cytokines and Chemokines in the Vitreous Fluid of Eyes with Uveal Melanoma. Invest. Ophthalmol. Vis. Sci. 2012;53(11):6748-6755. doi: 10.1167/iovs.12-10123.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: In uveal melanoma, both tumor size and an inflammatory phenotype have been shown to correlate with a poor clinical prognosis. The purpose of this study was to identify whether inflammatory cytokines were present in the vitreous of eyes with uveal melanoma and to determine whether the vitreal concentration of cytokines correlated with prognostic variables such as tumor dimensions and immune cell infiltrate.

Methods.: Vitreous was acquired from patients with uveal melanoma (n = 33) and from control eyes with no known ocular conditions (n = 9), and analyzed using a 27-plex cytokine bead array. Immunofluorescence testing was performed to determine the presence of macrophages, CD4+, CD8+, and Foxp3+ regulatory T cells (Tregs).

Results.: Compared with control eyes, eyes with uveal melanoma demonstrated higher vitreal concentrations of many cytokines and chemokines, including IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNF-α, and RANTES. IL-1ra was decreased in the vitreous of tumor-bearing eyes compared with controls. Tumor prominence positively correlated with several cytokines, including IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNF-α, RANTES, GCSF, IFN-γ, and VEGF. IL-6 and IP-10 were found to positively correlate with increasing regulatory T-cell infiltrate and IL-6 alone positively correlated with macrophage infiltration.

Conclusions.: Eyes with uveal melanoma contain higher vitreal concentrations of several inflammatory cytokines and chemokines that correlate predominantly with increasing tumor size; elevations in certain cytokines and chemokines also correlate with the presence of immune cell infiltrate.

Introduction
Uveal melanoma is the most common malignant primary intraocular tumor in adults and has a yearly incidence between 4.3 and 10.9 cases per million. 13 The tumor is derived from melanocytes in the uveal tract, consisting of the choroid, ciliary body, and iris. Treatments for uveal melanoma include enucleation and different modalities of irradiation such as plaque radiotherapy, stereotactic radiotherapy, and proton beam treatment. 4,5 However, despite the existence of these treatment modalities, up to 50% of patients develop metastatic disease, leading to death in an average of 8 to 10 months. Therefore, with the finding that uveal melanomas contain many immune cells, 69 immunotherapy may prove to be a useful treatment option; thus, better characterization of the tumor environment and the related immune components is important. 
A link between cancer and inflammation was described by Virchow in 1863 and since then, many studies have confirmed this connection between chronic inflammation and tumor progression. 10,11 Chronic inflammation has been shown to enhance tumor growth, invasion, and spread. 11 However, what determines whether immune cells will be pro- or antitumorigenic remains poorly understood. Moreover, a solid, malignant tumor consists of myriad cells (tumor cells, immune cells, fibroblasts, and endothelial cells), all of which contribute ultimately to tumor growth or regression. The activity and interactions of these cells result in the release of different soluble factors that provide valuable crosstalk and may lead to the potential recruitment of other cells to the tumor site. 
In models of other types of malignancies, including non-small cell lung cancer, gastric cancer, and cervical cancer, it has been demonstrated that chronic inflammation can be triggered by inflammatory mediators, including TNF-α, IL-6, and IL-17, all of which contribute to increased tumor growth and inhibition of antitumor activity. 12 Already antibodies against soluble mediators, such as bevacizumab (directed against vascular endothelial growth factor [VEGF]) or antibodies that compete with a soluble mediator, such as cetuximab (directed against epidermal growth factor receptor), are being used clinically as a therapy against cancer. Thus, characterizing these soluble mediators in the context of uveal melanoma represents an important goal in both developing a better understanding of this condition and a potential treatment. 
Several studies have evaluated the presence of cytokines in the anterior chamber and have demonstrated elevated concentrations of cytokines in the aqueous of eyes with uveal melanoma, 13 which do not seem to be predictive for survival. 14,15 However, a majority of uveal melanomas arise in the choroid and are bathed by the vitreous of the posterior eye. Moreover, in contrast to aqueous humor, which is constantly being replaced and replenished by the ciliary body, the vitreous is more stagnant and, thus, may better represent the chronic state of the eye. Interestingly, another study recently also evaluated cytokines in the vitreous of eyes with uveal melanoma, demonstrating elevations in several cytokines that generally correlated with levels in aqueous humor and tumor size. 15  
Our study aimed at further evaluating if cytokines are indeed elevated in eyes with uveal melanoma; in addition to evaluating correlations between tumor dimensions and cytokine levels, we have examined whether there is a relationship between the presence of immune cell infiltrate in the tumor and cytokine concentrations in the vitreous. 
Materials and Methods
Patient/Control Eyes
Vitreous was obtained from 33 eyes with uveal melanoma and 9 eyes with no known ocular conditions (controls). None of the eyes with uveal melanoma received treatment prior to enucleation. The eyes with uveal melanoma were acquired following enucleation between 1999 and 2004 and patient information/survival data were obtained from patient charts and updated to March 2012. Patients were informed about the potential use of their eyes for research purposes and signed informed consent forms prior to the enucleation. All procedures were performed according to guidelines agreed on by the Medical Ethics Committee at Leiden University Medical Center (Leiden, The Netherlands) and are consistent with principles established by the latest revision of the Declaration of Helsinki (World Medical Association Declaration of Helsinki; ethical principles for medical research involving human subjects). Immediately after enucleation of eyes with uveal melanoma, approximately 0.5 mL of vitreous was extracted. Control eyes were received from the Euro Tissue Bank (Beverwijk, The Netherlands) at 4°C and vitreous was extracted within 24 hours of the patient's demise. All samples of vitreous were stored at −80°C. 
Pathologic Analysis of Tumors
Following retrieval of vitreous, eyes were fixed in 4% formaldehyde solution for 48 hours and embedded in paraffin. Sections (4 μm) were cut, stained with hematoxylin and eosin, and reviewed by a pathologist for confirmation of diagnosis and to determine tumor characteristics: largest basal diameter, tumor prominence, cell type, and intraocular localization, including involvement of the ciliary body as well as scleral invasion, were recorded. 
Measurement of Cytokines and Chemokines in Vitreous
Samples of vitreous were thawed on ice and spun down at 10,000g for 5 minutes at 4°C. Vitreous was diluted 1:2 in 1% bovine serum albumin in phosphate-buffered saline (BSA/PBS), so that the final concentration of BSA was 0.5% and 50 μL of the diluted sample was pipetted into 96-well plates. A 27-plex cytokine bead array (Bio-Plex Pro Human Cytokine 27-plex Assay; Bio-Rad Laboratories, Veendendaal, The Netherlands) was used according to the manufacturer's instructions and samples were analyzed (Bio-Plex 200 system; Bio-Rad). The kit enables detection of the following cytokines and chemokines: IL-1b, IL-1rα, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17A, basic FGF, eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, and VEGF. 
Fluorescent Immunostaining of Tumor-Infiltrating Leukocytes
Phenotypic characterization of leukocytes had been performed using fluorescent immunostaining, as described previously. 16,17 Briefly, to visualize the subtypes of lymphocytes, deparaffinized and citrate antigen retrieval-treated 4-μm formalin-fixed sections were stained by a mixture of antibodies: CD3 (ab828; Abcam, Cambridge, UK), CD8 (4B11; Novocastra Antibodies/Leica Microsystems, Wetzlar, Germany), and the anti-Foxp3 antibody (clone 236A/E7; Abcam). 18 T helper cells were counted by identifying cells that were CD3+CD8Foxp3 (CD4+), a technique that has been described previously. 19 Macrophages in these tumors were identified using immunofluorescence staining with monoclonal antibodies directed against CD68 (clone 514H12; Abcam). Images were captured with a confocal laser scanning microscope (CLSM510; Carl Zeiss Meditec, Jena, Germany; with a PH2 Plan-NEOFluar ×25/0.80 Imm Korr objective; Carl Zeiss Meditec). Ten images were scanned per slide in high-power (×250) fields, and each scan represented one square optical field (area, 0.137 mm2). Positive lymphocytes were enumerated in these randomly selected fields. The amount of macrophage staining was objectively determined in pixels per mm2 by an image-analysis software program (Stacks; Department of Molecular Cell Biology, LUMC, Leiden, The Netherlands). Counts of intratumoral infiltrating leukocytes were represented per mm2
Chromosome 3 Status
Analysis of chromosome 3 status (both standard cytogenetic analysis and fluorescence in situ hybridization on isolated nuclei) of this patient material was described previously by Maat et al. 20  
Statistics
The Shapiro–Wilk test was used to test the normality of the data; because the data were not normally distributed, nonparametric statistical analysis was performed. Spearman's rank correlation coefficient was used to analyze correlations between numerical data, whereas the Mann–Whitney test was used to compare cytokine levels between two groups. Statistics were performed using commercial statistic software programs (SPSS 17; SPSS, Inc., Chicago, IL; and GraphPad Prism5; GraphPad Software Inc., La Jolla, CA) and P < .05 was considered statistically significant. 
Results
Cytokine Levels in Vitreous
Vitreous was collected from 33 eyes with uveal melanoma (Table 1) and 9 control eyes postenucleation. We first wanted to test whether the vitreous of eyes with uveal melanoma differs in the levels of proinflammatory cytokines from eyes without uveal melanoma. Of the 27 cytokines and chemokines screened, higher concentrations of IL-6 (P = 0.01), IL-8 (P = 0.004), IP-10 (P < 0.001), MCP-1 (P < 0.001), MIP-1α (P = 0.009), MIP-1β (P = 0.007), RANTES (P = .004), and TNF-α (P = 0.03) were observed in the vitreous of eyes with uveal melanoma when compared with that of the controls (Fig. 1, Table 2). In contrast, vitreous from eyes with uveal melanoma (UM) had lower concentrations of IL-1ra (P < 0.0001) compared with control vitreous (Fig. 1, Table 2). 
Figure 1. 
 
Cytokine levels in vitreous. Expression of nine cytokines and chemokines (pg/mL) in vitreous of eyes with uveal melanoma (tumor, n = 33) versus control eyes (n = 9). Horizontal line indicates the median in each group.
Figure 1. 
 
Cytokine levels in vitreous. Expression of nine cytokines and chemokines (pg/mL) in vitreous of eyes with uveal melanoma (tumor, n = 33) versus control eyes (n = 9). Horizontal line indicates the median in each group.
Table 1. 
 
Patient and Tumor Characteristics
Table 1. 
 
Patient and Tumor Characteristics
Characteristic Value
Age, y ± SEM 57.9 ± 2.6
Sex
 Male, n (%) 17 (51.5)
 Female, n (%) 16 (48.5)
Eye
 Right, n (%) 18 (54.5)
 Left, n (%) 15 (45.5)
Largest basal diameter, mm ± SD 13.1 ± 2.6
Prominence, mm ± SD  7.4 ± 2.1
TNM staging
 T1, n (%) 2 (6)
 T2, n (%) 12 (36)
 T3, n (%) 19 (58)
 T4, n (%) 0 (0)
Cell type
 Spindle, n (%) 7 (21)
 Epithelioid/mixed, n (%) 26 (79)
Chromosome 3 status
 Disomy, n (%) 12 (36)
 Monosomy, n (%) 21 (64)
Metastasis
 No, n (%) 20 (61)
 Yes, n (%) 13 (39)
5-year survival
 Alive, n (%) 20 (61)
 Deceased, n (%) 13 (39)
Table 2. 
 
Concentrations in Vitreous of Control Eyes and Eyes with Uveal Melanoma
Table 2. 
 
Concentrations in Vitreous of Control Eyes and Eyes with Uveal Melanoma
Cytokine Median (Range), pg/mL Significance
Eotaxin
 Control 0.92 (0.92–60.0) P = 0.90
 Uveal melanoma 0.92 (0.92–28.5)
FGF
 Control 120 (16.3–696) P = 0.43
 Uveal melanoma 37.6 (0.43–1,713)
GCSF
 Control 3.6 (0.16–41.6) P = 0.06
 Uveal melanoma 22.4 (0.12–113)
IFN-γ
 Control 2.6 (1.9–375) P = 0.14
 Uveal melanoma 34.0 (1.9–292)
IL-1b
 Control 0.25 (0.25–1.16) P = 0.51
 Uveal melanoma 0.25 (0.03–23.6)
IL-1ra
 Control 328 (132–1,056) P < 0.0001
 Uveal melanoma 38.5 (2.5–924)
IL-4
 Control 0.15 (0.05–1.5) P = 0.16
 Uveal melanoma 0.26 (0.00–2.56)
IL-5
 Control 0.19 (0.19–2.4) P = 0.67
 Uveal melanoma 0.19 (0.12–3.6)
IL-6
 Control 16.96 (3.18–117) P = 0.01
 Uveal melanoma 83.28 (0.17–265)
IL-7
 Control 31.3 (15.1–62.7) P = 0.24
 Uveal melanoma 25.9 (0.71–91.5)
IL-8
 Control 7.9 (4.88–99.4) P = 0.004
 Uveal melanoma 43.5 (1.03–256)
IL-10
 Control 0.71 (0.15–86.8) P = 0.53
 Uveal melanoma 0.71 (0.40–21.4)
IL-12
 Control 1.02 (1.02–316) P = 0.59
 Uveal melanoma 1.02 (0.14–92.3)
IL-13
 Control 0.22 (0.16–9.80) P = 0.32
 Uveal melanoma 0.85 (0.14–6.5)
IL-15
 Control 0.18 (0.01–2.04) P = 0.51
 Uveal melanoma 0.18 (0.08–5.9)
IP-10
 Control 2,535 (721–4614) P < 0.001
 Uveal melanoma 9,181 (17.13–12,078)
MCP
 Control 243 (141–463) P < 0.001
 Uveal melanoma 496 (10.3–540)
MIP-1α
 Control 0.59 (0.59–3.4) P = 0.009
 Uveal melanoma 2.2 (0.56–37.6)
MIP-1β
 Control 3.7 (0.51–14.9) P = 0.007
 Uveal melanoma 13.1 (0.51–111)
PDGF
 Control 0.65 (0.65–101) P = 0.86
 Uveal melanoma 0.65 (0.65–31.8)
RANTES
 Control 4.6 (0.64–44.3) P = 0.004
 Uveal melanoma 29.7 (0.64–381)
TNF-α
 Control 0.42 (0.42–26.8) P = 0.03
 Uveal melanoma 6.3 (0.42–31.1)
VEGF
 Control 2.5 (2.5–29.4) P = 0.15
 Uveal melanoma 4.7 (1.9–1,527)
No significant difference was observed between VEGF levels in control versus tumor eyes. IL-2, IL-9, IL-17, and GM-CSF levels were below the detection threshold of the assay. A significant difference in age was observed between the group with UM and the control, 57.9 ± 2.63 (mean age ± SEM) and 72.2 ± 3.24, respectively. Linear regression analysis was performed to determine whether age, rather than the presence of tumor, contributed to the level of cytokine expression and no significant effect of age was demonstrated. 
Cytokine Concentration and Tumor Prominence
Vitreous from tumor-bearing eyes generally had higher cytokine concentrations than that of control eyes; however, in the tumor-bearing group we included patients with a range of tumor sizes. Therefore, we also sought to compare within the uveal melanoma group whether tumor size correlated with cytokine levels. Interestingly, when we compared within the tumor group, we found additional cytokines that correlated with tumor size. The following cytokines positively correlated with increasing tumor prominence: IL-6 (P = 0.04), IL-8 (P = 0.02), IP-10 (P < 0.001), MIP-1α (P = 0.002), MIP-1β (P = 0.03), GCSF (P = 0.007), IFN-γ (P = 0.01), RANTES (P = 0.02), TNF-α (P = 0.048), and VEGF (P = 0.008) (Fig. 2). No statistically significant negative correlations were observed between cytokine levels and tumor prominence. Both TNF-α (r = −0.418, P = 0.015) and MIP-1α (r = −0.369, P = 0.03) negatively correlated with largest basal diameter of the tumor; however, there were no statistically significant positive correlations between cytokine concentration and largest basal diameter. Also, no correlations between cytokine concentrations and tumor cell morphology (spindle, epithelioid, or mixed) or with chromosome 3 status were found (data not shown). 
Figure 2. 
 
Cytokine and chemokine concentration and tumor prominence. Positive correlations between concentration (pg/mL) of cytokines and chemokines in the vitreous of eyes with uveal melanoma (n = 33) and tumor prominence (mm). Spearman's correlation coefficient (r) shows strength of correlation and P < 0.05 was considered significant.
Figure 2. 
 
Cytokine and chemokine concentration and tumor prominence. Positive correlations between concentration (pg/mL) of cytokines and chemokines in the vitreous of eyes with uveal melanoma (n = 33) and tumor prominence (mm). Spearman's correlation coefficient (r) shows strength of correlation and P < 0.05 was considered significant.
Correlations of Cytokine Concentrations with Immune Cell Infiltrate
Although the eye is an immune-privileged site, both macrophages and T cells have been found in uveal melanoma and the presence of a leukocytic infiltrate is associated with a poor clinical prognosis. 68 We hypothesized that tumors with more immune infiltrate would have higher concentrations of cytokines in the vitreous. We stained sections to determine the presence of macrophages, cytotoxic T cells, and regulatory T cells (Tregs) using antibodies for CD68, CD8, and Foxp3, respectively. The presence of CD4+ T helper cells was determined by identification of cells that were CD3+CD8Foxp3. The total amount of positive staining was measured and for each marker, tumors were stratified into two groups based on whether the quantity of staining was above or below the median. Of the 27 cytokines and chemokines screened, IL-6 was the only cytokine that was significantly different between tumors with high CD68 and low CD68 staining and was significantly higher in tumors with greater CD68 staining (P = 0.03) (Fig. 3). Both IP-10 (P = 0.03) and IL-6 (P = 0.02) concentrations were higher in tumors that contained greater Foxp3+ staining (Fig. 3, Table 3). There were no observed differences in cytokine concentrations between tumors with high versus low amounts of T helper or cytotoxic T-cell infiltrate. Furthermore, immune cell infiltrate did not correlate with tumor prominence. 
Figure 3. 
 
Correlations of cytokine and chemokine concentrations with immune cell infiltrate. Expression of cytokines and chemokines (pg/mL) in the vitreous of uveal melanoma eyes with high versus low tumor infiltration by CD68+ cells (top) and Foxp3+ cells (bottom).
Figure 3. 
 
Correlations of cytokine and chemokine concentrations with immune cell infiltrate. Expression of cytokines and chemokines (pg/mL) in the vitreous of uveal melanoma eyes with high versus low tumor infiltration by CD68+ cells (top) and Foxp3+ cells (bottom).
Table 3. 
 
IL-6 and IP-10 Concentration and Immune Cell Infiltrate
Table 3. 
 
IL-6 and IP-10 Concentration and Immune Cell Infiltrate
Cytokine Immune Cell Staining Median (Range), pg/mL Significance
IL-6 Low CD68+ 48.1 (0.17–265) P = 0.03
High CD68+ 101.6 (25.7–200)
Low Foxp3+ 46.1 (0.17–265) P = 0.02
High Foxp3+ 101.6 (25.7–200)
IP-10 Low CD68+ 9,024 (17.1–11,712) P = 0.38
High CD68+ 9,615 (1,999–12,078)
Low Foxp3+ 7,815 (17–11,436) P = 0.03
High Foxp3+ 10,692 (1,999–12,078)
Factors Predictive of Vitreal IL-6 and IP-10 Concentrations
Because IL-6 was related to tumor prominence as well as the degree of infiltration by both macrophages and Foxp3+ cells, we performed binary logistic regression to determine which of these three factors was most predictive of IL-6 concentrations in the vitreous. We also used this statistical analysis to determine whether Foxp3+ infiltrate or prominence was more predictive of IP-10 levels in the vitreous since these two factors independently correlated with IP-10 concentrations. The presence of CD68+ cells in the tumor was more predictive of IL-6 levels than the prominence of Foxp3+ infiltrate. For IP-10, prominence was more predictive of vitreal levels than the amount of Foxp3+ infiltrate. 
Clustering of Cytokines and Chemokines
We also performed hierarchical clustering analysis to ascertain which cytokines and chemokines grouped together in eyes with uveal melanoma. Cytokines such as IL-1b and IL-1ra or MIP-1α and MIP-1β cluster together in uveal melanoma, which is interesting because these are groups of cytokines that generally show correlations in other models as well. The dendrogram in Supplemental Figure S1 (see Supplementary Material and Supplementary Fig. S1) highlights other clusters that we found in the vitreous of eyes with uveal melanoma. 
Predictive Value of Vitreal Cytokine Concentrations for Survival
Because tumor prominence and the presence of an inflammatory phenotype have both been linked to poor prognosis, we studied whether the concentration of cytokines or chemokines in the vitreous was related to the length of survival postenucleation. Patients were stratified into two groups based on whether the concentration of a cytokine was above or below the median and Kaplan–Meier curves were made. Statistically, only IL-13 affected survival with higher concentrations of IL-13 associated with greater survival. However, in our sample group, the levels of IL-13 fell within a narrow range of 0.14 to 6.49 pg/mL, with a median concentration of 0.85 pg/mL. No other cytokine significantly affected survival. 
Discussion
The present analysis has demonstrated increases in vitreal concentrations of eight biomarkers (IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNF-α, and RANTES) in the vitreous of eyes with uveal melanoma compared with eyes without and makes the novel finding that vitreal concentrations of IL-1ra were decreased in tumor eyes compared with controls. Although Dunavoelgyi et al. 15 reported that in eyes with uveal melanoma, six cytokines correlate with tumor dimensions, we demonstrate that increasing tumor prominence correlates with elevations in IL-6, IL-8, IP-10, MIP-1α, MIP-1β, GCSF, IFN-γ, RANTES, TNF-α, and VEGF. The present study also demonstrates a relationship between immune cell infiltrate and cytokine levels. Elevations in IL-6 and IP-10 correlated with increased Treg infiltration and IL-6 alone positively correlated with increased macrophage infiltration of the tumor. Finally, despite increases in inflammatory cytokines in eyes with uveal melanoma, only IL-13 levels predicted survival statistically. 
IL-6 appears to play a significant role in uveal melanoma; in our study, elevations in this cytokine correlated with both increased tumor prominence and the presence of both macrophage and Treg infiltration of the tumor. Elevations in vitreal concentrations of IL-6 in uveal melanoma were also observed in another study 15 ; however, the correlation between IL-6 and immune infiltrate represents a new finding worthy of further discussion. One in vitro study demonstrated that conditioned media from both uveal melanoma and monocyte cell lines that do not independently produce IL-6 could trigger IL-6 production by the other cell line. 21 This study suggests IL-6 may play a critical role in the crosstalk between uveal melanoma cells and macrophages; however, the factor responsible for triggering these cells to produce IL-6 remains unknown. 
The finding that increased IL-6 levels correlate with increased Treg infiltration of the tumor requires further investigation into the specific pathways responsible for this phenomenon. IL-6 has been shown to inhibit T-cell differentiation into Tregs. 22 Thus, it may appear contradictory that there would be a positive correlation between the two. One hypothesis is that the increased IL-6 may be a host response to tumor growth, released to inhibit differentiation of T cells into Tregs with immunosuppressive properties. However, Eikawa et al. 23 demonstrated that tumor cell lines, including one melanoma cell line, that produce IL-6 induce marked migration of Tregs. Moreover, other studies have demonstrated that IL-6, under certain inflammatory conditions in vivo, can trigger Foxp3+ T-cell induction. 24 These studies support an alternative hypothesis that elevated IL-6 production by the tumor may lead to increased Treg migration to the tumor environment. We also observed that elevations in vitreal concentrations of IP-10 correlated with increased Treg infiltration of the tumor. In other models of inflammatory conditions, including atherosclerosis and sarcoidosis, IP-10 secretion recruits Tregs to affected organs and increases the activity of these cells. 25,26  
In our findings, IL-1ra was significantly decreased in the vitreous of uveal melanoma patients when compared with controls. This suggests a protective role for IL-1ra and decreased levels may be associated with tumor progression. IL-1ra has been more thoroughly explored in other tumors 27 and pathologies wherein it has been demonstrated to block IL-1 signaling. In rheumatoid arthritis, synovial fluid has elevated levels of IL-1 thought to be involved in joint destruction and neovascularization 28,29 ; therefore, recombinant IL-1ra has been approved for treatment of this condition in both the United States and Europe. In lung and melanoma cancer models, IL-1 has been shown to be required for tumor invasiveness and angiogenesis. 27 It is hypothesized that IL-1ra may act by inhibition of inflammation and neovascularization; thus, these properties may also be of use against tumors. In cutaneous melanoma, IL-1ra has been shown in vivo to decrease growth and metastatic spread of tumor. 30 Few studies have examined the role of IL-1ra in uveal melanoma and, interestingly, the role of IL-1 is controversial, with studies demonstrating both antitumor 31 and protumor effects. 32 One study in a murine model demonstrated that inhibition of IL-1 with IL-1ra not only decreased uveal melanoma tumor growth, but also modulated both immune response and tumor stroma. 32 In our study, it can also be speculated that, since IL-1 levels were not significantly affected despite changes in IL-1ra, IL-1ra may have a function distinct from the effects it mediates through IL-1. Our findings support the idea that exploration of IL-1ra for the treatment of uveal melanoma may be of great value. 
Recent therapeutic strategies targeted at inhibition of uveal melanoma growth include the use of anti-VEGF. Interestingly, whereas some studies have shown an increase in VEGF concentration in the aqueous or vitreous of eyes with uveal melanoma when compared with controls, 14,33 others have found no significant difference. 13 In our study, similarly to that published by Lee et al., 13 who examined VEGF in aqueous, we found VEGF was generally more highly expressed in eyes with uveal melanoma than those without. However, in both studies, this observation was not statistically significant. In our study, only 57% of vitreous from tumor eyes had detectable VEGF; those eyes with measurable VEGF did have high concentrations of it. A previous study by our laboratory demonstrated that VEGF concentrations are elevated in eyes with uveal melanoma if there is serous retinal detachment 34 ; thus, this variable may explain the discrepancies between these various studies on VEGF levels in ocular fluids. 
In uveal melanoma, monosomy 3 is associated with an inflammatory phenotype of the tumor 35,36 and poor clinical prognosis. 37,38 We did not find an association between monosomy 3 and elevations in inflammatory cytokines. This may be due to our sample size or the fact that in uveal melanoma, tumor size also significantly affects prognosis. 39,40 In addition, we found no relationship between tumor prominence and degree of immune infiltration. In uveal melanoma, greater tumor diameter or height is associated with increased mortality rates: 16% for small tumors (<3 mm height and <10 mm diameter), 32% for medium tumors (3–8 mm in height and <15 mm diameter), and 53% for large tumors (>8 mm in height and >15 mm diameter). 39,41 One study demonstrated that each millimeter increment of uveal melanoma growth was associated with a significant increase in metastatic potential, with tumors >7 mm associated with more than a 40% chance of metastasis. 42 Our sample population is already skewed toward larger tumors (average prominence was 7.5 mm in our study) because smaller tumors generally do not require enucleation of the eye. The preselection of large tumors may also explain why we generally failed to see correlations between cytokine level and survival. 
The one cytokine wherein increased vitreous concentrations statistically correlated with improved survival was IL-13; however, this cytokine was present in a narrow range. Although little is known regarding the role of IL-13 in uveal melanoma, several studies in other cancer models have proposed interesting roles for this cytokine in cancer therapy. IL-13, a member of the TH2 family of cytokines, with significant homology to IL-4, has been shown to exhibit certain anticancer effects independent of the IL-4 receptor. 43 Overexpression of the IL-13 receptor (IL-13R) has been demonstrated on many tumor cells from Kaposi's sarcoma, malignant glioma cells, renal cell carcinoma, and squamous cell carcinoma of the head and neck; interestingly, several of these tumors are sensitive to treatment with IL-13. 4447 Whether the IL-13R is present on uveal melanoma cells has yet to be determined. IL-13 is also interesting in that it is produced by NK cells and in animal models of uveal melanoma, the presence of NK cells reduces the potential formation of micrometastases. 48,49 Therefore, we hypothesize that even slight increases in IL-13 concentration may increase NK cell activity and lead to decreased metastatic outcomes and greater survival. 
In conclusion, myriad soluble factors are released in response to uveal melanoma growth. These factors not only may contribute directly to tumor progression but also may affect the immune response. Further characterization of the components responsible for increases in cytokines may lead to greater insight into the pathophysiology of this complex condition and may enable direct targeting of certain cells or mediators to inhibit tumor growth or enhance immune response. 
Supplementary Materials
Acknowledgments
The authors thank Mieke Versluis, PhD, for her assistance in the collection of patient samples and helpful discussions; Geert Haasnoot, statistician, for help with the statistical analysis; and Els van Beelen for the use of the Bio-Plex 200 system. 
References
Virgili G Gatta G Ciccolallo L Incidence of uveal melanoma in Europe. Ophthalmology . 2007;114:2309–2315. [CrossRef] [PubMed]
Singh AD Bergman L Seregard S. Uveal melanoma: epidemiologic aspects. Ophthalmol Clin North Am . 2005;18:75–84, viii. [CrossRef] [PubMed]
Singh AD Topham A. Incidence of uveal melanoma in the United States: 1973–1997. Ophthalmology . 2003;110:956–961. [CrossRef] [PubMed]
Jager MJ Niederkorn JY Ksander BR eds. Uveal Melanoma: A Model for Exploring Fundamental Cancer Biology . London: Taylor & Francis; 2004.
Desjardins L Lumbroso-Le Rouic L Levy-Gabriel C Combined proton beam radiotherapy and transpupillary thermotherapy for large uveal melanomas: a randomized study of 151 patients. Ophthalmic Res . 2006;38:255–260. [CrossRef] [PubMed]
Makitie T Summanen P Tarkkanen A Kivela T. Tumor-infiltrating macrophages (CD68(+) cells) and prognosis in malignant uveal melanoma. Invest Ophthalmol Vis Sci . 2001;42:1414–1421. [PubMed]
Durie FH Campbell AM Lee WR Damato BE. Analysis of lymphocytic infiltration in uveal melanoma. Invest Ophthalmol Vis Sci . 1990;31:2106–2110. [PubMed]
de la Cruz PO Jr Specht CS McLean IW. Lymphocytic infiltration in uveal malignant melanoma. Cancer . 1990;65:112–115. [CrossRef] [PubMed]
Toivonen P Makitie T Kujala E Kivela T. Microcirculation and tumor-infiltrating macrophages in choroidal and ciliary body melanoma and corresponding metastases. Invest Ophthalmol Vis Sci . 2004;45:1–6. [CrossRef] [PubMed]
Balkwill F Mantovani A. Inflammation and cancer: back to Virchow? Lancet . 2001;357:539–545. [CrossRef] [PubMed]
Coussens LM Werb Z. Inflammation and cancer. Nature . 2002;420:860–867. [CrossRef] [PubMed]
Lin WW Karin M. A cytokine-mediated link between innate immunity, inflammation, and cancer. J Clin Invest . 2007;117:1175–1183. [CrossRef] [PubMed]
Lee CS Jun IH Kim TI Byeon SH Koh HJ Lee SC. Expression of 12 cytokines in aqueous humour of uveal melanoma before and after combined Ruthenium-106 brachytherapy and transpupillary thermotherapy. Acta Ophthalmol . 2012;90:e314–e320. [CrossRef] [PubMed]
Ly LV Bronkhorst IH van Beelen E Inflammatory cytokines in eyes with uveal melanoma and relation with macrophage infiltration. Invest Ophthalmol Vis Sci . 2010;51:5445–5451. [CrossRef] [PubMed]
Dunavoelgyi R Funk M Sacu S Intraocular activation of angiogenic and inflammatory pathways in uveal melanoma. Retina . 2012;32:1373–1384. [CrossRef] [PubMed]
Bronkhorst IH Ly LV Jordanova ES Detection of M2-macrophages in uveal melanoma and relation with survival. Invest Ophthalmol Vis Sci . 2011;52:643–650. [CrossRef] [PubMed]
van Vlierberghe RL Sandel MH Prins FA Four-color staining combining fluorescence and brightfield microscopy for simultaneous immune cell phenotyping and localization in tumor tissue sections. Microsc Res Tech . 2005;67:15–21. [CrossRef] [PubMed]
Bronkhorst IHG, Khanh Vu TH, Jordanova ES, Luyten GPM, van der Burg SH, Jager MJ. Different subsets of tumor-infiltrating lymphocytes correlate with macrophage influx and monosomy 3 in uveal melanoma. Invest Ophthalmol Vis Sci . 2012;53:5370–5378. [CrossRef] [PubMed]
Piersma SJ Jordanova ES van Poelgeest MI High number of intraepithelial CD8+ tumor-infiltrating lymphocytes is associated with the absence of lymph node metastases in patients with large early-stage cervical cancer. Cancer Res . 2007;67:354–361. [CrossRef] [PubMed]
Maat W Jordanova ES van Zelderen-Bhola SL The heterogeneous distribution of monosomy 3 in uveal melanomas: implications for prognostication based on fine-needle aspiration biopsies. Arch Pathol Lab Med . 2007;131:91–96. [PubMed]
Cools-Lartigue J Marshall JC Caissie AL Saraiva VS Burnier MN Jr. Secretion of interleukin-6 and prostaglandin E2 during uveal melanoma-monocyte in vitro interactions. Exp Eye Res . 2004;79:451–454. [CrossRef] [PubMed]
Kishimoto T. IL-6: from its discovery to clinical applications. Int Immunol . 2010;22:347–352. [CrossRef] [PubMed]
Eikawa S Ohue Y Kitaoka K Enrichment of Foxp3+ CD4 regulatory T cells in migrated T cells to IL-6- and IL-8-expressing tumors through predominant induction of CXCR1 by IL-6. J Immunol . 2010;185:6734–6740. [CrossRef] [PubMed]
Nakagawa T Tsuruoka M Ogura H IL-6 positively regulates Foxp3+CD8+ T cells in vivo. Int Immunol . 2010;22:129–139. [CrossRef] [PubMed]
Miyara M Amoura Z Parizot C The immune paradox of sarcoidosis and regulatory T cells. J Exp Med . 2006;203:359–370. [CrossRef] [PubMed]
Heller EA Liu E Tager AM Chemokine CXCL10 promotes atherogenesis by modulating the local balance of effector and regulatory T cells. Circulation . 2006;113:2301–2312. [CrossRef] [PubMed]
Voronov E Shouval DS Krelin Y IL-1 is required for tumor invasiveness and angiogenesis. Proc Natl Acad Sci USA . 2003;100:2645–2650. [CrossRef] [PubMed]
Feldmann M Maini RN. The role of cytokines in the pathogenesis of rheumatoid arthritis. Rheumatology (Oxford) . 1999;38 (suppl 2);3–7. [PubMed]
Maruotti N Cantatore FP Crivellato E Vacca A Ribatti D. Angiogenesis in rheumatoid arthritis. Histol Histopathol . 2006;21:557–566. [PubMed]
Weinreich DM Elaraj DM Puhlmann M Effect of interleukin 1 receptor antagonist gene transduction on human melanoma xenografts in nude mice. Cancer Res . 2003;63:5957–5961. [PubMed]
Woodward JK Elshaw SR Murray AK Stimulation and inhibition of uveal melanoma invasion by HGF, GRO, IL-1alpha, and TGF-beta. Invest Ophthalmol Vis Sci . 2002;43:3144–3152. [PubMed]
Triozzi PL Aldrich W Singh A. Effects of interleukin-1 receptor antagonist on tumor stroma in experimental uveal melanoma. Invest Ophthalmol Vis Sci . 2011;52:5529–5535. [CrossRef] [PubMed]
Boyd SR Tan D Bunce C Vascular endothelial growth factor is elevated in ocular fluids of eyes harbouring uveal melanoma: identification of a potential therapeutic window. Br J Ophthalmol . 2002;86:448–452. [CrossRef] [PubMed]
Missotten GS Notting IC Schlingemann RO Vascular endothelial growth factor A in eyes with uveal melanoma. Arch Ophthalmol . 2006;124:1428–1434. [CrossRef] [PubMed]
Bronkhorst IHG Jager MJ. Uveal melanoma: the inflammatory microenvironment. J Innate Immun . 2012;4:454–462. [CrossRef] [PubMed]
Maat W Ly LV Jordanova ES de Wolff-Rouendaal D Schalij-Delfos NE Jager MJ. Monosomy of chromosome 3 and an inflammatory phenotype occur together in uveal melanoma. Invest Ophthalmol Vis Sci . 2008;49:505–510. [CrossRef] [PubMed]
van den Bosch T van Beek JG Vaarwater J Higher percentage of FISH-determined monosomy 3 and 8q amplification in uveal melanoma cells relate to poor patient prognosis. Invest Ophthalmol Vis Sci . 2012;53:2668–2674. [CrossRef] [PubMed]
Bronkhorst IH Maat W Jordanova ES Effect of heterogeneous distribution of monosomy 3 on prognosis in uveal melanoma. Arch Pathol Lab Med . 2011;135:1042–1047. [CrossRef] [PubMed]
Singh AD Shields CL Shields JA. Prognostic factors in uveal melanoma. Melanoma Res . 2001;11:255–263. [CrossRef] [PubMed]
Damato B Coupland SE. A reappraisal of the significance of largest basal diameter of posterior uveal melanoma. Eye . 2009;23:2152–2160 ; quiz 2161–2162. [CrossRef] [PubMed]
Diener-West M Hawkins BS Markowitz JA Schachat AP. A review of mortality from choroidal melanoma. II. A meta-analysis of 5-year mortality rates following enucleation, 1966 through 1988. Arch Ophthalmol . 1992;110:245–250. [CrossRef] [PubMed]
Shields CL Furuta M Thangappan A Metastasis of uveal melanoma millimeter-by-millimeter in 8033 consecutive eyes. Arch Ophthalmol . 2009;127:989–998. [CrossRef] [PubMed]
Obiri NI Husain SR Debinski W Puri RK. Interleukin 13 inhibits growth of human renal cell carcinoma cells independently of the p140 interleukin 4 receptor chain. Clin Cancer Res . 1996;2:1743–1749. [PubMed]
Debinski W Obiri NI Powers SK Pastan I Puri RK. Human glioma cells overexpress receptors for interleukin 13 and are extremely sensitive to a novel chimeric protein composed of interleukin 13 and pseudomonas exotoxin. Clin Cancer Res . 1995;1:1253–1258. [PubMed]
Husain SR Obiri NI Gill P Receptor for interleukin 13 on AIDS-associated Kaposi's sarcoma cells serves as a new target for a potent Pseudomonas exotoxin-based chimeric toxin protein. Clin Cancer Res . 1997;3:151–156. [PubMed]
Kawakami K Kawakami M Joshi BH Puri RK. Interleukin-13 receptor-targeted cancer therapy in an immunodeficient animal model of human head and neck cancer. Cancer Res . 2001;61:6194–6200. [PubMed]
Kawakami K Kioi M Liu Q Kawakami M Puri RK. Evidence that IL-13R alpha2 chain in human glioma cells is responsible for the antitumor activity mediated by receptor-directed cytotoxin therapy. J Immunother . 2005;28:193–202. [CrossRef] [PubMed]
Yang H Dithmar S Grossniklaus HE. Interferon alpha 2b decreases hepatic micrometastasis in a murine model of ocular melanoma by activation of intrinsic hepatic natural killer cells. Invest Ophthalmol Vis Sci . 2004;45:2056–2064. [CrossRef] [PubMed]
Dithmar SA Rusciano DA Armstrong CA Lynn MJ Grossniklaus HE. Depletion of NK cell activity results in growth of hepatic micrometastases in a murine ocular melanoma model. Curr Eye Res . 1999;19:426–431. [CrossRef] [PubMed]
Footnotes
 Supported in part by UitZicht (NN-G and MJJ), Stichting Nederlands Oogheelkundig Onderzoek, Gelderse Blinden Stichting, Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, Stichting Blinden-Penning, Landelijke Stichting voor Blinden en Slechtzienden, and an MD/PhD Trajectory Grant from the Board of Directors of the Leiden University Medical Center (IHGB).
Footnotes
 Disclosure: N. Nagarkatti-Gude, None; I.H.G. Bronkhorst, None; S.G. van Duinen, None; G.P.M. Luyten, None; M.J. Jager, None
Figure 1. 
 
Cytokine levels in vitreous. Expression of nine cytokines and chemokines (pg/mL) in vitreous of eyes with uveal melanoma (tumor, n = 33) versus control eyes (n = 9). Horizontal line indicates the median in each group.
Figure 1. 
 
Cytokine levels in vitreous. Expression of nine cytokines and chemokines (pg/mL) in vitreous of eyes with uveal melanoma (tumor, n = 33) versus control eyes (n = 9). Horizontal line indicates the median in each group.
Figure 2. 
 
Cytokine and chemokine concentration and tumor prominence. Positive correlations between concentration (pg/mL) of cytokines and chemokines in the vitreous of eyes with uveal melanoma (n = 33) and tumor prominence (mm). Spearman's correlation coefficient (r) shows strength of correlation and P < 0.05 was considered significant.
Figure 2. 
 
Cytokine and chemokine concentration and tumor prominence. Positive correlations between concentration (pg/mL) of cytokines and chemokines in the vitreous of eyes with uveal melanoma (n = 33) and tumor prominence (mm). Spearman's correlation coefficient (r) shows strength of correlation and P < 0.05 was considered significant.
Figure 3. 
 
Correlations of cytokine and chemokine concentrations with immune cell infiltrate. Expression of cytokines and chemokines (pg/mL) in the vitreous of uveal melanoma eyes with high versus low tumor infiltration by CD68+ cells (top) and Foxp3+ cells (bottom).
Figure 3. 
 
Correlations of cytokine and chemokine concentrations with immune cell infiltrate. Expression of cytokines and chemokines (pg/mL) in the vitreous of uveal melanoma eyes with high versus low tumor infiltration by CD68+ cells (top) and Foxp3+ cells (bottom).
Table 1. 
 
Patient and Tumor Characteristics
Table 1. 
 
Patient and Tumor Characteristics
Characteristic Value
Age, y ± SEM 57.9 ± 2.6
Sex
 Male, n (%) 17 (51.5)
 Female, n (%) 16 (48.5)
Eye
 Right, n (%) 18 (54.5)
 Left, n (%) 15 (45.5)
Largest basal diameter, mm ± SD 13.1 ± 2.6
Prominence, mm ± SD  7.4 ± 2.1
TNM staging
 T1, n (%) 2 (6)
 T2, n (%) 12 (36)
 T3, n (%) 19 (58)
 T4, n (%) 0 (0)
Cell type
 Spindle, n (%) 7 (21)
 Epithelioid/mixed, n (%) 26 (79)
Chromosome 3 status
 Disomy, n (%) 12 (36)
 Monosomy, n (%) 21 (64)
Metastasis
 No, n (%) 20 (61)
 Yes, n (%) 13 (39)
5-year survival
 Alive, n (%) 20 (61)
 Deceased, n (%) 13 (39)
Table 2. 
 
Concentrations in Vitreous of Control Eyes and Eyes with Uveal Melanoma
Table 2. 
 
Concentrations in Vitreous of Control Eyes and Eyes with Uveal Melanoma
Cytokine Median (Range), pg/mL Significance
Eotaxin
 Control 0.92 (0.92–60.0) P = 0.90
 Uveal melanoma 0.92 (0.92–28.5)
FGF
 Control 120 (16.3–696) P = 0.43
 Uveal melanoma 37.6 (0.43–1,713)
GCSF
 Control 3.6 (0.16–41.6) P = 0.06
 Uveal melanoma 22.4 (0.12–113)
IFN-γ
 Control 2.6 (1.9–375) P = 0.14
 Uveal melanoma 34.0 (1.9–292)
IL-1b
 Control 0.25 (0.25–1.16) P = 0.51
 Uveal melanoma 0.25 (0.03–23.6)
IL-1ra
 Control 328 (132–1,056) P < 0.0001
 Uveal melanoma 38.5 (2.5–924)
IL-4
 Control 0.15 (0.05–1.5) P = 0.16
 Uveal melanoma 0.26 (0.00–2.56)
IL-5
 Control 0.19 (0.19–2.4) P = 0.67
 Uveal melanoma 0.19 (0.12–3.6)
IL-6
 Control 16.96 (3.18–117) P = 0.01
 Uveal melanoma 83.28 (0.17–265)
IL-7
 Control 31.3 (15.1–62.7) P = 0.24
 Uveal melanoma 25.9 (0.71–91.5)
IL-8
 Control 7.9 (4.88–99.4) P = 0.004
 Uveal melanoma 43.5 (1.03–256)
IL-10
 Control 0.71 (0.15–86.8) P = 0.53
 Uveal melanoma 0.71 (0.40–21.4)
IL-12
 Control 1.02 (1.02–316) P = 0.59
 Uveal melanoma 1.02 (0.14–92.3)
IL-13
 Control 0.22 (0.16–9.80) P = 0.32
 Uveal melanoma 0.85 (0.14–6.5)
IL-15
 Control 0.18 (0.01–2.04) P = 0.51
 Uveal melanoma 0.18 (0.08–5.9)
IP-10
 Control 2,535 (721–4614) P < 0.001
 Uveal melanoma 9,181 (17.13–12,078)
MCP
 Control 243 (141–463) P < 0.001
 Uveal melanoma 496 (10.3–540)
MIP-1α
 Control 0.59 (0.59–3.4) P = 0.009
 Uveal melanoma 2.2 (0.56–37.6)
MIP-1β
 Control 3.7 (0.51–14.9) P = 0.007
 Uveal melanoma 13.1 (0.51–111)
PDGF
 Control 0.65 (0.65–101) P = 0.86
 Uveal melanoma 0.65 (0.65–31.8)
RANTES
 Control 4.6 (0.64–44.3) P = 0.004
 Uveal melanoma 29.7 (0.64–381)
TNF-α
 Control 0.42 (0.42–26.8) P = 0.03
 Uveal melanoma 6.3 (0.42–31.1)
VEGF
 Control 2.5 (2.5–29.4) P = 0.15
 Uveal melanoma 4.7 (1.9–1,527)
Table 3. 
 
IL-6 and IP-10 Concentration and Immune Cell Infiltrate
Table 3. 
 
IL-6 and IP-10 Concentration and Immune Cell Infiltrate
Cytokine Immune Cell Staining Median (Range), pg/mL Significance
IL-6 Low CD68+ 48.1 (0.17–265) P = 0.03
High CD68+ 101.6 (25.7–200)
Low Foxp3+ 46.1 (0.17–265) P = 0.02
High Foxp3+ 101.6 (25.7–200)
IP-10 Low CD68+ 9,024 (17.1–11,712) P = 0.38
High CD68+ 9,615 (1,999–12,078)
Low Foxp3+ 7,815 (17–11,436) P = 0.03
High Foxp3+ 10,692 (1,999–12,078)
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