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Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75235
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-melanocyte-stimulating hormone, glucocorticoids,
and TGF-ß (7, 8, 9, 10, 11). Fas ligand is expressed throughout
the eye and is responsible for the induction of apoptosis in
lymphocytes bearing its receptor, Fas (12). Recognition of
intraocular target cells by T cells is impaired by low expression
levels of MHC class I and II on ocular tissues (13). When
Ag recognition does take place in the anterior chamber, a deviant
immune response ensues, which down-regulates delayed-type
hypersensitivity and preserves the integrity of delicate,
nonregenerating ocular tissues (2, 3). The innate response is also suppressed in the eye. We have recently demonstrated that the pleiotropic cytokine, macrophage migration-inhibitory factor (MIF),3 is present in the aqueous humor and produces an immediate inhibition of NK cell-mediated cytolysis in vitro (14, 15). The presence of MIF in the aqueous humor appears to protect intraocular tumors against NK cell-mediated surveillance, given that tumors that undergo NK cell-mediated rejection at extraocular sites grow progressively in the eye (16). Considerable evidence indicates that NK cells are important in controlling metastases of intraocular melanomas. In vivo depletion of NK cells with anti-asialo-GM1 Ab results in a sharp increase in metastases in mice bearing intraocular melanomas (17). Moreover, human uveal melanoma cells displaying high levels of MHC class I molecules are resistant to NK cell-mediated lysis in vitro and produce more extensive metastases than melanoma cells expressing low MHC class I (17).
MIF is produced by numerous cells in the eye including the lens, iris, ciliary body, retinal pigment epithelial cells, Müller cells, and corneal endothelial cells (18, 19, 20, 21). Moreover, uveal melanomas arise from ocular tissues that produce MIF (19) and are often directly exposed to the aqueous humor, which contains biologically relevant concentrations of MIF (14, 15). Within the eye, one would expect uveal melanomas to be protected from NK cell-mediated immune surveillance. However, once uveal melanomas metastasize to the liver, an organ with exceptionally high levels of inherent NK activity (22, 23), the melanomas would be vulnerable to NK cell-mediated elimination. Accordingly, we suspected that uveal melanomas might produce MIF as a means of protecting themselves against NK cells following metastasis from the eye.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were obtained from the mouse colony at the University of Texas Southwestern Medical Center, Dallas, TX. All animals were treated in accordance with the guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Tumor cell lines
Uveal melanoma cell lines Mel 202, Mel 270, Mel 290, and OMM 2.3 were generously provided by Dr. Bruce Ksander, Schepens Eye Research Institute, Boston, MA (24). OMM2.3 is derived from a liver metastasis that originated from the primary tumor represented by the Mel 270 cell line. We thank Dr. June Kan-Mitchell, University of California, San Diego, CA, for OCM1, OCM3, and OCM8 uveal melanoma cells and Dr. Gregorius P. Luyten, University Hospital Rotterdam, Rotterdam, The Netherlands, for OMM1 cells which were isolated from a s.c. metastasis originating from a uveal melanoma (25, 26, 27). Dr. Martine Jager, Leiden University Hospital, Leiden, The Netherlands, kindly provided 92-1 cells (28). YAC-1 murine lymphoma cells were a gift from Dr. Michael Bennett (University of Texas Southwestern Medical Center). OCM1 and OCM3 cells were maintained in Ham’s F-12 medium (BioWhittaker, Walkersville, MD) containing 10% heat-inactivated FBS (HyClone Laboratories, Logan, UT), 2 mM L-glutamine (JRH Biosciences, Lenexa, KS), 10 mM HEPES buffer solution (JRH Biosciences), 1% nonessential amino acids solution (BioWhittaker), 1% penicillin-streptomycin-Fungizone solution (BioWhittaker), and 1 mM sodium pyruvate (JRH Biosciences). Mel 202, Mel 270, Mel 290, OMM 2.3, OCM8, OMM1, 92-1, and YAC-1 cells were maintained in complete RPMI 1640 (JRH Biosciences) containing the same additives as the Ham’s F-12 with the addition of 5 x 10-5 M 2-ME (Sigma, St. Louis, MO).
MIF ELISA
Uveal melanoma supernatants were assayed for MIF by sandwich ELISA. Plates were coated overnight with a mouse IgG anti-human MIF capture Ab (R&D Systems, Minneapolis, MN) at 2 µg/ml and blocked for 3 h with PBS containing 1% BSA and 5% sucrose. Uveal melanoma supernatants were added for a 2-h incubation, followed by detection with a goat IgG anti-human MIF Ab (R&D Systems) at 0.2 µg/ml for 2 h. The detection reaction was completed by addition of streptavidin-HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) followed by 1 mg/ml 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) with 0.003% H2O2. Plates were read at 405 nm, and readings from uveal melanoma supernatants were compared with a standard curve of recombinant MIF readings to give concentrations of MIF.
Inhibition of macrophage migration
Macrophage migration assays were performed as previously described (29). Briefly, spleen cells from C57BL/6 mice were washed, overlaid on Histopaque 1083 (Sigma), and centrifuged for 20 min at 3000 rpm. The buffy layer cells were resuspended to 1 x 108 cells/ml and 40 µl of this suspension were drawn into glass capillary tubes capped at one end. Tubes were centrifuged at 1500 rpm for 5 min, cut at the interface of the pellet and supernatant, and placed horizontally in 24-well plates. Wells were filled with 2x complete RPMI, 2x uveal melanoma supernatant, or aqueous humor, and macrophages were incubated for 24 h at 37°C, 5% CO2 to allow macrophage migration from the capillary tube into the well. The migration area was calculated after 24 h.
NK cell cytotoxicity assay
NK cell-mediated cytolysis of tumor targets was assessed using a conventional 4-h 51Cr release assay previously described (30). Lymphokine-activated killer (LAK) cells used as effectors were obtained from C57BL/6 mice. LAK cells have been previously used in place of resting NK cells for a variety of in vitro and in vivo experiments because they give higher and more consistent levels of cytolysis than resting NK cells. Moreover, LAK cells demonstrate the same in vivo effector function as NK cells (31). Briefly, spleens were collected, erythrocytes were lysed, and splenocytes were washed with HBSS (BioWhittaker) before 4 days of culture at 37°C, 10% CO2 in complete DMEM medium (JRH Biosciences) containing 10% heat-inactivated FBS (HyClone Laboratories), 2 mM L-glutamine (JRH Biosciences), 1 mM sodium pyruvate (JRH Biosciences), 2 mM MEM vitamins (JRH Biosciences), 1% penicillin-streptomycin-Fungizone solution (BioWhittaker), 5 x 10-5 M 2-ME (Sigma), and 1000 U/ml recombinant human IL-2 (Hoffman-La Roche, Nutley, NJ). This procedure has previously been shown to enrich for NK cells (32). After 4 days of culture, LAK cells were collected and centrifuged over Histopaque 1083 (Sigma) at 3000 rpm for 20 min. The buffy layer was collected, washed, and used as a source of effector cells in cytotoxicity assays where either YAC-1 cells or human uveal melanoma cells were used as targets.
Inhibition of NK cell-mediated killing
NK-mediated lysis was inhibited by incubating LAK cells with rabbit aqueous humor, or uveal melanoma supernatants for 30 min before combining the LAK effector cells with target cells. Aqueous humor was harvested from the anterior chamber of rabbit eyeballs (Pel-Freez Biologicals, Rogers, AR) by paracentesis under sterile conditions as previously described (14). Uveal melanoma supernatants were obtained from 3-day cultures of 5 x 106 uveal melanoma cells in 10 ml medium.
Neutralization of MIF
To demonstrate that the observed inhibition of NK lysis was mediated by MIF, we pre-treated four uveal melanoma supernatants with a goat anti-human MIF Ab (R&D Systems) or a control goat IgG Ab (Sigma) at a concentration of 67 µg/ml for 15 min. After this Ab treatment, supernatants were used to treat LAK cells, which were then used as effectors as described above. Dose dependency of the anti-MIF Ab was shown by treating OMM2.3 supernatant with anti-MIF Ab concentrations ranging from 600 to 7 µg/ml for 15 min and then proceeding with LAK treatment and target cell lysis as above.
Statistics
Differences between groups were analyzed by Student’s t test; p values <0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As an additional negative control to demonstrate that the MIF Ab was
not binding nonspecifically to the plate, we tested blank wells
containing no MIF Ag. All cell lines expressed MIF, and levels of MIF
expression were highest in cell lines OMM1 and OMM2.3, both of which
were isolated from uveal melanoma metastases (Fig. 1). Elevated MIF
secretion by both metastatic cell lines supports the hypothesis that a
uveal melanoma can create its own microenvironment of immune privilege
once it leaves the immune privileged ocular site.
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Our ELISA results confirmed the presence of secreted MIF in that
the anti-MIF Ab bound a specific MIF epitope, but an ELISA assay
cannot show a protein to be active. To determine whether the MIF
detected by ELISA was biologically active, uveal melanoma supernatants
were tested in a bioassay. MIF was originally named for its ability to
inhibit the migration of macrophages, and the standard bioassay for MIF
activity remains the most sensitive one for detecting this cytokine
(29). Accordingly, the biological activity of the
MIF-containing uveal melanoma supernatants was tested in a macrophage
migration inhibition assay using rabbit aqueous humor as a positive
control (15) (Fig. 2).
Aqueous humor containing MIF inhibited macrophage migration by 
70%.
The negative control used was 2x RPMI, relevant because all melanoma
supernatants were concentrated 2-fold before being used in this assay.
Uveal melanoma supernatants from Mel 270 and OMM2.3 cell lines
significantly inhibited the migration of macrophages out of capillary
tubes as compared with the medium control (Fig. 2, A–D).
Supernatant from Mel 270 cells caused 40% inhibition of migration,
and OMM2.3 supernatant slowed migration 50%, as shown in Fig. 2E.
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Previously, it has been shown that recombinant MIF and aqueous
humor-derived MIF inhibit NK cell-mediated lysis of YAC-1 lymphoma
target cells (15). Therefore, we hypothesized that the
biologically active MIF found in uveal melanoma supernatants would
inhibit NK-mediated cytolysis of YAC-1 cells. The ability of
MIF-containing cell supernatants to inhibit YAC-1 cell lysis by LAK
cells was tested in a 4-h 51Cr release assay. LAK
cells were incubated in supernatants before adding YAC-1 target cells
to allow MIF in the supernatants to have an effect on the LAK cells,
and supernatants continued to be included throughout the 4-h incubation
(Fig. 3). Killing of YAC-1 cell targets
was 70% in untreated wells containing RPMI and did not change
significantly when 2x RPMI was included, demonstrating that a 2-fold
concentration of medium did not impact the ability of LAK cells to kill
YAC-1 targets. This negative control was necessary because all uveal
melanoma supernatants were concentrated 2-fold before their use in this
assay. As a positive control, LAK cells were incubated in aqueous
humor, which resulted in a decreased killing level of 50%
(15). All uveal melanoma cell lines tested significantly
inhibited killing of YAC-1 cells by LAK cells compared with the 2x
RPMI control; levels of killing in the presence of uveal melanoma
supernatants ranged from 30 to 70%.
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The first group included cell lines that secreted high amounts of MIF
by ELISA and, as expected, strongly inhibited NK killing. The second
group contained cell lines that secreted little MIF and produced only
weak inhibition of killing. The third was composed of cell lines that
secreted low amounts of MIF yet still inhibited NK killing. Abrogation of NK inhibition by anti-MIF Ab
To determine whether the observed NK inhibition was due to the
action of MIF, uveal melanoma supernatants were incubated with
anti-MIF Ab before the supernatants were added to LAK cells (Fig. 4A). Supernatants from four
uveal melanoma cell lines were treated with either anti-MIF Ab or
an isotype control goat IgG Ab. The inhibitory activity of OMM2.3 and
OMM1 supernatants, both containing high amounts of MIF, was
significantly neutralized by anti-MIF Ab but was unaffected by the
control Ab. By contrast, Mel 270 and Mel 290 supernatants, both of
which contain little MIF, were not significantly affected by
anti-MIF or control Ab.
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B). Untreated supernatant from OMM2.3 cells was included as
a negative control in which MIF in OMM2.3 supernatant protected YAC-1
target cells and very little killing took place. Similarly, OMM2.3
supernatant treated with control Ab showed minimal lysis. A maximal
dose of 200 µg/ml anti-MIF Ab neutralized MIF in the OMM2.3
supernatant and produced 40% YAC-1 killing. As the dose of
anti-MIF Ab decreased from 200 to 7 µg/ml, the abrogation of NK
inhibition lessened accordingly. MIF-containing supernatant protects uveal melanomas from lysis
As a control experiment to demonstrate that human uveal melanomas
are indeed susceptible to NK-mediated cytolysis, human uveal melanoma
cells in the absence of conditioned melanoma supernatant were exposed
to LAK effectors in a 4-h 51Cr release assay.
Without the protection of secreted MIF found in conditioned
supernatant, all 10 uveal melanoma cell lines tested were susceptible
to killing, to varying degrees (Fig. 5).
YAC-1 cells were included as highly NK-susceptible positive control
targets and were lysed at a rate of 75% in this experiment. Percent
lysis for human uveal melanoma cell lines ranged from 28 to 57%,
suggesting that uveal melanoma cells themselves are not inherently
resistant to NK killing and might benefit from inhibition of NK killing
by MIF.
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).
Target cells and supernatants were tested in various combinations to
determine whether the level of secreted MIF correlated with the level
of NK protection afforded. The results show that Mel 270 and OMM2.3
supernatant furnished a significant amount of protection against NK
killing for both Mel 270 and OMM2.3 target cells. Although OMM2.3
secreted 2-fold more MIF in ELISA assays, no significant difference
was found between levels of NK protection furnished by OMM2.3 and Mel
270 supernatants.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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If NK cells are an integral part of antitumor defense, an unexplained issue was how NK-susceptible uveal melanomas manage to evade NK-mediated destruction outside of the NK-quenching ocular environment to form metastases in the liver and in other sites. Lysis by NK cells is down-regulated within the eye, because MIF found in aqueous humor has been shown to inhibit NK killing of YAC-1 murine lymphoma, RMA-S, and uveal melanoma target cells (14, 15, 16). The present results show that the NK-inhibiting immune privilege of aqueous humor is perpetuated by uveal melanoma cells, which produce bioactive MIF that protects them from NK cytolysis.
MIF is produced in several extraocular tissues including brain, kidney, skin, lung, uterus, and liver (34, 35, 36, 37, 38), but it is not known whether it abrogates NK function in those sites. Despite its expression of MIF, the liver is known as a site of strong NK activity (22, 23). Uveal melanoma metastasizes preferentially to the liver, where its ability to secrete MIF may protect it from killing by NK cells. We hypothesize that uveal melanoma-produced MIF is involved in creating a microenvironment surrounding the melanoma cells in which MIF is found at a higher concentration than that found throughout the liver. This environment of concentrated MIF may provide immune privilege and protect the tumor cells from NK-mediated lysis.
Two groups of uveal melanoma cell lines provided supportive evidence
for our hypothesis that tumor-secreted MIF protected targets from NK
killing (Fig. 3). The first group included metastatic cell lines that
produced high levels of secreted MIF and strongly inhibited LAK
effectors from lysing YAC-1 targets. A second group of primary uveal
melanoma cell lines secreted low amounts of MIF and only weakly limited
the lysis of YAC-1 cells. A third group of uveal melanoma supernatants
inhibited NK killing despite secreting very little MIF. Additionally,
although anti-MIF Ab significantly decreased the protective effect
of supernatants from the first group containing MIF, it did not inhibit
completely (Fig. 4A). These two points suggest that another
NK-inhibiting factor may be secreted by uveal melanoma cells in
addition to MIF. One candidate factor is TGF-ß, which is known to
down-regulate NK activity. However, TGF-B is not likely to be at work
in these 4 h assays because it requires a much longer, 20-h
incubation period to exert NK-inhibiting effects (39).
IL-10 and PGE2 may be responsible for the NK
inhibition not attributable to MIF, or it may be that a novel
NK-inhibiting factor is secreted by uveal melanoma cells (39, 40).
Our results showing that metastases express elevated levels of MIF correlate with the work of Meyer-Siegler and Hudson (41), who showed by differential display PCR that MIF expression increases during the progression of prostate adenocarcinoma. Their work showed elevated MIF expression in focal carcinoma lesions as compared with normal prostate tissue, and a further augmentation of expression in metastatic prostate tissue harvested from lymph nodes. Our work, in addition to showing that MIF protein is secreted at a greater level by uveal melanoma metastases as compared with primary tumors, assigns a function to MIF as a protector of tumor cells from NK elimination. Whether the increased production of MIF by metastases is the result of selective pressure exerted by NK cells or is an intrinsic property of melanocytes as they undergo malignant transformation remains unknown. However, the recent findings of Shimizu et al. (42) are particularly illuminating. They found that normal skin melanocytes express MIF mRNA, however MIF expression was much higher in melanoma cells and correlated with tumor progression. Moreover, MIF expression correlated with increased melanoma cell proliferation, migration, and tumor-induced angiogenesis. Our findings and those of Shimizu et al. (42) suggest that the pleiotropic properties of MIF may contribute to the growth and metastasis of skin and ocular melanomas by affecting multiple parameters, both immunological and nonimmunological.
These data provide evidence for a mechanism by which NK-susceptible uveal melanoma tumors survive outside of the immune-privileged environment of the eye. The results suggest that uveal melanomas create their own NK-inhibiting immune privilege by secreting bioactive MIF, which inhibits NK-mediated cytolysis of uveal melanoma target cells. Because uveal melanoma preferentially metastasizes to the liver where NK function is particularly active, secretion of MIF by uveal melanoma cells may provide an immune privilege crucial for metastatic growth.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jerry Y. Niederkorn, Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9057.
3 Abbreviations used in this paper: MIF, macrophage migration-inhibitory factor; LAK, lymphokine-activated NK cell.
Received for publication January 18, 2000. Accepted for publication April 25, 2000.
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References |
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/B produced by Kupffer cells inhibits interleukin-1, tumor necrosis factor production and interleukin-2-induced activation of nonparenchymal cells. Cancer Immunol. Immunother. 34:150.[Medline]
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