Effects of Interleukin-1 Receptor Antagonist on Tumor
Stroma in Experimental Uveal Melanoma
Pierre L. Triozzi, Wayne Aldrich, and Arun Singh
PURPOSE. In contrast to many malignancies showing evidence
that interleukin-1 (IL-1) promotes progression through effects
on tumor vascularity and myeloid suppressor cell populations,
in uveal melanoma there is evidence that IL-1 can inhibit
METHODS. The effects of the IL-1 receptor antagonist IL-1ra
against the aggressive/invasive MUM2B and the nonaggressive/
noninvasive OCM1 uveal melanoma models were examined in
vitro and in vivo in mouse xenografts. Vascularity and myeloid
suppressor cell populations and their regulators were assessed.
RESULTS. In vitro, IL-1, and IL-1ra did not affect the proliferation
of the uveal melanoma cells or their production of IL-1, IL-6,
transforming growth factor (TGF) ?, or VEGF. In vivo, IL-1ra
treatment resulted in substantial growth inhibition of MUM2B
tumors; less inhibition was observed against OCM1 tumors.
Periodic acid-Schiff loops and CD11b?macrophages within
the tumor stroma decreased in vivo; CD31?blood vessels were
not altered. IL-1ra treatment in vivo did not affect tumor-
derived IL-1, IL-6, TGF-?, or VEGF. In contrast, host IL-1?, IL-6,
and tumor necrosis factor decreased. Host VEGF was not al-
tered. Intratumoral IL-12(p40) and CXCL10, markers of host
M1 polarization, increased, and intratumoral arginase and
CD206, markers of myeloid-derived suppressor cells (MDSC)
and M2 macrophage polarization, decreased. IL-1ra treatment
in vivo also reduced splenic CD11b?Gr1?MDSC.
CONCLUSIONS. IL-1 may play a role in promoting uveal melanoma
progression. Inhibiting IL-1 with IL-1ra inhibits tumor growth
in vivo but not in vitro. Tumor stroma is modified, myeloid
suppressor cells are reduced, and M1 macrophage polarization
is increased in vivo. (Invest Ophthalmol Vis Sci. 2011;52:
poor; the median survival is approximately 6 months. Chemo-
therapeutics used to treat cutaneous melanoma rarely produce
durable responses in patients with uveal melanoma, and new
systemic treatments are needed.1Interleukin (IL)-1, an endog-
enous mediator of acute and chronic inflammatory conditions,
has demonstrated antitumor activity, including the ability to
enhance antitumor immune responses and chemotherapy cy-
totoxicity and to inhibit tumor migration and invasion.2–4
etastatic disease will develop in as many as 40% of pa-
tients with uveal melanoma, the prognosis of which is
There is accumulating evidence, however, that both IL-1 iso-
forms (IL-1? and IL-1?) may play roles in tumor development,
invasion, angiogenesis, and metastases, either directly or indi-
rectly through the induction of other cytokines (see Ref. 5 for
review). IL-1 may also contribute to the ability of tumors to
escape immune surveillance by promoting M2-polarized mac-
rophages and myeloid-derived suppressor cells (MDSC).6–9
Although several studies support inhibiting IL-1 in cancer
therapy, little information is available regarding the role of IL-1
in uveal melanoma. During inflammatory responses in the eye,
IL-1 is produced by macrophages and corneal cells and pro-
motes a number of processes that may be tumor promoting,
including angiogenesis.10Among the transcripts found to be
upregulated in the progression from intraocular to metastatic
uveal melanoma was that of the IL-1 receptor.11There is also
evidence that providing IL-1 may be therapeutic in uveal mel-
anoma. IL-1 has been shown to inhibit uveal melanoma migra-
tion and invasion.4Gene expression for the IL-1 receptor ac-
cessory protein essential for IL-1 signaling is underexpressed in
uveal melanoma manifesting monosomy 3, which is associated
with metastasis.12Moreover, there is evidence that IL-1 also
does not promote, but rather abrogates, the immune-privileged
nature of the ocular environment implicated in protecting
uveal melanoma from destruction by immune effectors.13
A naturally occurring IL-1 antagonist, IL-1ra, competitively
blocks IL-1? and IL-1? at the receptor level. A human recom-
binant IL-1ra is used clinically to treat patients with rheumatoid
arthritis, and its application to many ocular inflammatory dis-
eases is also under investigation.14–16Human recombinant
IL-1ra has been shown to inhibit the development and growth
of metastases in several animal tumor models, including mouse
B16 melanoma and human cutaneous melanoma xeno-
grafts.9,17–20Anti-inflammatory drugs are being tested to lessen
the toxicity of plaque radiotherapy in patients with uveal
melanoma.21Given this and the potential contradictory roles
of IL-1, we examined the effects of IL-1ra in two established
MUM2B model and the poorly invasive/aggressive OCM1
model (see Ref. 22 for review). We focused on the modulation
of tumor vascularity and myeloid suppressor cell populations.
MATERIALS AND METHODS
Cell Lines and Animals
MUM2B and OCM1 human uveal melanoma cell lines were maintained
in Dulbecco’s modified Essential medium (DMEM) with 10% heat-
inactivated fetal calf serum, 1 mM sodium pyruvate, 100 U/mL peni-
cillin, and 100 ?g/mL streptomycin (Mediatech, Herndon, VA).23The
cultures were grown at 37°C in 5% CO2to confluence, passaged by
treatment with 0.05% trypsin in EDTA at 37°C, and washed in media
before being centrifuged at 200g for 10 minutes to form a pellet.
Athymic NCR male nu/nu mice 4 to 6 weeks of age were obtained
from Taconic Farms (Hudson, NY) and were fed a commercial diet and
water ad libitum. All experimental procedures on the animals were
performed according to the ARVO Statement for the Use of Animals in
From the Cleveland Clinic Taussig Cancer Institute, Cleveland,
Supported in part by National Institutes of Health Grant
Submitted for publication August 3, 2010; revised December 3,
2010, January 12, 2011, and March 8, 2011; accepted March 9, 2011.
Disclosure: P.L. Triozzi, None; W. Aldrich, None; A. Singh,
Corresponding author: Pierre L. Triozzi, Cleveland Clinic Taussig
Cancer Institute, R40, 9500 Euclid Avenue, Cleveland, OH 44195;
Immunology and Microbiology
Investigative Ophthalmology & Visual Science, July 2011, Vol. 52, No. 8
Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc.
29. Mills CD, Shearer J, Evans R, Caldwell MD. Macrophage arginine
metabolism and the inhibition or stimulation of cancer. J Immu-
30. Umemura N, Saio M, Suwa T, et al. Tumor-infiltrating myeloid-
derived suppressor cells are pleiotropic-inflamed monocytes/mac-
rophages that bear M1- and M2-type characteristics. J Leukoc Biol.
31. Yurkovetsky ZR, Kirkwood JM, Edington HD, et al. Multiplex
analysis of serum cytokines in melanoma patients treated with
interferon-alpha2b. Clin Cancer Res. 2007;13:2422–2428.
32. Omulecki W, Damato BE, Sekundo W, Lee WR, Toczyska-Rozentryt
E, Omulecka A. Bilateral uveal melanoma presenting simultane-
ously. Ger J Ophthalmol. 1994;3:228–231.
33. Ijland SA, Jager MJ, Heijdra BM, Westphal JR, Peek R. Expression of
angiogenic and immunosuppressive factors by uveal melanoma
cell lines. Melanoma Res. 1999;9:445–450.
34. Yang H, Jager MJ, Grossniklaus HE. Bevacizumab suppression of
establishment of micrometastases in experimental ocular mela-
noma. Invest Ophthalmol Vis Sci. 2010;51:2835–2842.
35. Wolf JS, Chen Z, Dong G, et al. IL (interleukin)-1 promotes nuclear
factor-B and AP-1-induced IL-8 expression, cell survival, and pro-
liferation in head and neck squamous cell carcinomas. Clin Cancer
36. La E, Rundhaug JE, Fischer SM. Role of intracellular interleukin-1
receptor antagonist in skin carcinogenesis. Mol Carcinog. 2001;
37. Hsieh TC, Chiao JW. Growth modulation of human prostatic can-
cer cells by interleukin-1 and interleukin-1 receptor antagonist.
Cancer Lett. 1995;95:119–123.
38. Yamada Y, Karasaki H, Matsushima K, Lee GH, Ogawa K. Expres-
sion of an IL-1 receptor antagonist during mouse hepatocarcino-
genesis demonstrated by differential display analysis. Lab Invest.
39. Oelmann E, Kraemer A, Serve H, et al. Autocrine interleukin-1
receptor antagonist can support malignant growth of glioblastoma
by blocking growth-inhibiting autocrine loop of interleukin-1. Int
J Cancer. 1997;71:1066–1076.
40. El Filali M, Homminga I, Maat W, van der Velden PA, Jager MJ.
Triamcinolone acetonide and anecortave acetate do not stimulate
uveal melanoma cell growth. Mol Vis. 2008;14:1752–1759.
41. Huang JJ, Newton RC, Rutledge SJ, et al. Characterization of mu-
rine IL-1 beta: isolation, expression, and purification. J Immunol.
42. Libert C, Brouckaert P, Shaw A, Fiers W. Induction of interleukin
6 by human and murine recombinant interleukin 1 in mice. Eur
J Immunol. 1990;20:691–694.
43. Demou ZN, Hendrix MJ. Microgenomics profile the endogenous
angiogenic phenotype in subpopulations of aggressive melanoma.
J Cell Biochem. 2008;105:562–573.
44. Seftor RE, Seftor EA, Kirschmann DA, Hendrix MJ. Targeting the
tumor microenvironment with chemically modified tetracyclines:
inhibition of laminin 5 gamma2 chain promigratory fragments and
vasculogenic mimicry. Mol Cancer Ther. 2002;1:1173–1179.
45. Hess AR, Seftor EA, Seftor RE, Hendrix MJ. Phosphoinositide 3-ki-
nase regulates membrane Type 1-matrix metalloproteinase (MMP)
and MMP-2 activity during melanoma cell vasculogenic mimicry.
Cancer Res. 2003;63:4757–4762.
46. Zhang S, Li M, Gu Y, et al. Thalidomide influences growth and
vasculogenic mimicry channel formation in melanoma. J Exp Clin
Cancer Res. 2008;27:60.
47. Cong R, Sun Q, Yang L, Gu H, Zeng Y, Wang B. Effect of Genistein
on vasculogenic mimicry formation by human uveal melanoma
cells. J Exp Clin Cancer Res. 2009;28:124.
48. Basu GD, Pathangey LB, Tinder TL, Gendler SJ, Mukherjee P.
Mechanisms underlying the growth inhibitory effects of the cyclo-
oxygenase-2 inhibitor celecoxib in human breast cancer cells.
Breast Cancer Res. 2005;7:R422–R435.
49. de Waard-Siebinga I, Hilders CG, Hansen BE, van Delft JL, Jager MJ.
HLA expression and tumor-infiltrating immune cells in uveal mel-
anoma. Graefes Arch Clin Exp Ophthalmol. 1996;234:34–42.
50. 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.
51. 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.
52. McKenna KC, Kapp JA. Accumulation of immunosuppressive
CD11b? myeloid cells correlates with the failure to prevent tumor
growth in the anterior chamber of the eye. J Immunol. 2006;177:
53. Ly LV, Baghat A, Versluis M, et al. In aged mice, outgrowth of
intraocular melanoma depends on proangiogenic M2-type macro-
phages. J Immunol. 2010;185:3481–3488.
54. McKenna KC, Beatty KM, Bilonick RA, Schoenfield L, Lathrop KL,
Singh AD. Activated CD11b? CD15? granulocytes increase in the
blood of patients with uveal melanoma. Invest Ophthalmol Vis Sci.
55. Atochina O, Daly-Engel T, Piskorska D, McGuire E, Harn DA. A
schistosome-expressed immunomodulatory glycoconjugate ex-
pands peritoneal Gr1(?) macrophages that suppress naive
CD4(?) T cell proliferation via an IFN-gamma and nitric oxide-
dependent mechanism. J Immunol. 2001;167:4293–4302.
56. Dana MR, Zhu SN, Yamada J. Topical modulation of interleukin-1
activity in corneal neovascularization. Cornea. 1998;17:403–409.
57. Stapleton WM, Chaurasia SS, Medeiros FW, Mohan RR, Sinha S,
Wilson SE. Topical interleukin-1 receptor antagonist inhibits in-
flammatory cell infiltration into the cornea. Exp Eye Res. 2008;86:
58. Yamada J, Zhu SN, Streilein JW, Dana MR. Interleukin-1 receptor
antagonist therapy and induction of anterior chamber-associated
immune deviation-type tolerance after corneal transplantation. In-
vest Ophthalmol Vis Sci. 2000;41:4203–4208.
59. Marshall JC, Fernandes BF, Di Cesare S, et al. The use of a cyclo-
oxygenase-2 inhibitor (Nepafenac) in an ocular and metastatic
animal model of uveal melanoma. Carcinogenesis. 2007;28:2053–
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