Originally published online as doi:10.1189/jlb.1003487 on February 24, 2004
Published online before print February 24, 2004
(Journal of Leukocyte Biology. 2004;75:772-776.)
© 2004
by Society for Leukocyte Biology
Endothelial monocyte-activating polypeptide-II (EMAP-II): a novel inducer of lymphocyte apoptosis
J. C. Murray*,1,
Y. M. Heng*,
P. Symonds*,
K. Rice*,
W. Ward*,
M. Huggins
,
I. Todd and
R. A. Robins
* Wolfson Digestive Diseases Centre, University Hospital, and
Division of Immunology, School of Molecular Medical Sciences, University of Nottingham, United Kingdom
1 Correspondence: Wolfson Digestive Diseases Centre, University Hospital, University of Nottingham, Nottingham NG7 2UH, U.K. E-mail: cliff.murray{at}nott.ac.uk
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ABSTRACT
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The novel, proinflammatory cytokine endothelial monocyte-activating polypeptide-II (EMAP-II) was first found in tumor cell supernatants. EMAP-II is closely related or identical to the p43 auxiliary protein of the multisynthase complex, which is involved in protein synthesis. In vitro, EMAP-II induces procoagulant activity, increased expression of E- and P-selectins and tumor necrosis factor receptor-1, and ultimately, programmed cell death (apoptosis) in cultured endothelial cells. EMAP-II is also chemotactic for monocytes and neutrophils. However, the role of the p43/EMAP-II cytokine form in tumors is not understood. We hypothesized an immune-regulatory role within neoplastic tissues and investigated its effects on lymphocytes. EMAP-II causes a dose-dependent inhibition of proliferation and apoptosis in Jurkat T cells and mitogen-activated peripheral blood mononuclear cells. Coculture with DLD-1 colorectal cancer cells or media conditioned by these cells induces apoptosis in Jurkat cells, which is partially reversed by antibodies against EMAP-II. Our data suggest that EMAP-II constitutes a component of a novel, immunosuppressive pathway in solid tumors, which is not normally expressed outside the cell but in tumors, may be subject to abnormal processing and released from tumor cells.
Key Words: cytokine • tumor • p43 • immune evasion
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INTRODUCTION
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Endothelial monocyte-activating polypeptide-II (EMAP-II) was first detected in supernatants of cultured murine tumor cells (for reviews, see refs. [1
, 2
]) based on its ability to stimulate procoagulant activity in cultured endothelial cells. In 1992, Kao et al. [3
] isolated 20–22 kDa EMAP-II protein, which proved to have pleiotropic, cytokine-like activity toward endothelial cells as well as monocytes and neutrophils.
Full-length cDNAs encoding human EMAP-II have been isolated [4
, 5
]. The amino-terminal region of mature EMAP-II, where much of the cytokine activity resides [6
], has minor sequence homology with interleukin (IL)-1ß, IL-8, and von Willebrand factor antigen II, as well as structural homology with the chemokines monocyte chemoattractant protein-1 and regulated on activation, normal T expressed and secreted [7
]. The cDNA sequence described by Kao et al. [4
] is consistent with a 34-kDa precursor molecule of 328 amino acid residues, which is proteolytically cleaved at a critical aspartate residue (Asp144) to produce an 
18-kDa mature polypeptide. Behrensdorf et al. [8
] have shown that the 34-kDa precursor form is sensitive to cleavage by caspase 7, and some studies suggest that other proteases may be more important [9
, 10
]. As it lacks a classic hydrophobic signal peptide, the mature molecule may be secreted via a novel pathway; however, little is known about the mechanism of this processing or its control. EMAP-II may be processed in a similar manner to IL-1ß, which undergoes proteolytic cleavage at the plasma membrane with subsequent release into the extracellular space in the form of microvesicles [11
].
EMAP-II and the p43 auxiliary component of the mammalian multisynthetase complex share a high degree of amino acid identity [12
]. p43 shares 86% and 85% amino acid identity with human and murine 34 kDa EMAP-II, respectively, and the human p43 and EMAP-II homologues are identical [12
].
The 18- to 20-kDa form of EMAP-II possesses a wide range of activities in vitro, including the induction of tissue factor-dependent coagulation on endothelial cells and monocytes, up-regulation of endothelial E- and P-selectin expression, and release of von Willebrand factor [4
]. It is also chemotactic for neutrophils and monocytes and induces the release of myeloperoxidase from neutrophils [4
]. In vivo, injection of EMAP-II into the mouse footpad evokes an acute, inflammatory response characterized by edema and a neutrophil-rich infiltrate [3
]. Direct injection of EMAP-II into subcutaneous tumors in mice leads to hemorrhage and neutrophil-rich inflammatory infiltrates, followed by a decrease in tumor volume [4
]. EMAP-II has also been shown to induce endothelial apoptosis in vitro [13
, 14
], leading to speculation that its antitumor activity may be conferred in part by antiangiogenic activity, based on the induction of programmed cell death in endothelial cells. Furthermore, in vivo studies show that EMAP negatively regulates neovascularization and morphogenesis in the developing lung [15
].
Working on the principle that the secretion of the soluble form of EMAP-II may confer some biological benefit to the tumor, we have attempted to reconcile its chemotactic and apoptotic properties. If inflammatory cells are attracted into the tumor microenvironment to clear cellular debris, a mechanism to simultaneously inhibit the activity of tumor-specific T cells would be advantageous. We hypothesized that EMAP-II might therefore be toxic toward lymphocytes as well as endothelium and have tested this using cell-culture models of tumor cell–lymphocyte interactions.
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MATERIALS AND METHODS
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Cell lines
The human T cell line Jurkat and the colorectal adenocarcinoma cell line DLD-1 were obtained from American Type Culture Collection (Manassas, VA) and were cultured in RPMI-1640 medium (Gibco, UK) supplemented with 10% fetal calf serum (PAA Laboratories, Lintz, Austria) and 100 U/ml penicillin/streptomycin solution (Sigma, UK). Cells were maintained at 37°C in 5% CO2 in a humidified incubator and were routinely subcultured by removal from flasks with 0.05% trypsin/1 mM EDTA (Sigma).
Rapid amplification of 5'-cDNA ends (5' RACE) mapping of transcription start sites
Amplifications were performed on FirstChoice RACE-Ready cDNA prepared from normal human testis (Ambion, TX) according to protocol. This cDNA sample was manufactured such that the majority of full-length, decapped mRNA acquires an adaptor sequence at its "true" 5' end. Briefly, 1 µl cDNA was used for primary polymerase chain reaction (PCR) using an outer EMAP-II-specific antisense primer, designated EII10R (5'-CTCAAAGTTGCCTGCAAAATTGC-3'), and 5' RACE outer primer provided in the kit. Secondary amplification was performed on 1/50th of the primary PCR reaction using an inner primer, designated EII9R (5'-CTCCTTAAGTAGAGAAACTTGC-3'), and 5' RACE inner primer provided. Both EMAP-II-specific primers were designed from within the conserved exon 2 to amplify any potential, alternative first exons. All PCR reactions were performed using Taq polymerase and buffer supplied by Boehringer (UK) in a 50-µl vol under the following conditions: 94°C, 3 min (94°C, 1 min; 61°C, 1 min; 72°C, 2 min) x 35 cycles; 72°C, 5 min. PCR products were cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA) and sequenced by automated sequencing (Lark Technologies, UK).
Antibodies and recombinant proteins
Rabbit polyclonal antibodies against human EMAP-II (R2B2) were used for immunohistochemistry, Western blotting, and flow cytometry in this study [5
, 9
, 16
]. Horseradish peroxidase (HRP)-labeled anti-rabbit antibodies (Sigma) were used for detection of primary antibodies. Recombinant human tumor necrosis factor 
(rhTNF-) and interferon-
(IFN-) were obtained from PeproTech (UK). rhEMAP-II was generated in-house as described [5
] or purchased from PeproTech.
Detection of EMAP-II by Western blotting
Western blotting was performed essentially as described [5
]. Cells were trypsinized and washed with cold phosphate-buffered saline (PBS), and counted 5 x 106 cells were lysed in 400 µl sodium dodecyl sulfate (SDS) buffer (0.5 M Tris/HCl, 10% glycerol, 10% w/v SDS, 2% ß-mercaptoethanol, 0.1% bromophenol blue). Supernatant proteins were precipitated with ice-cold acetone and resuspended in 400 µl SDS buffer. Samples were boiled for 10 min, electrophoresed by SDS-polyacrylamide gel electrophoresis on 12% gels, and transferred onto nitrocellulose membranes (Amersham, UK). Membranes were blocked overnight with 5% nonfat dried milk in 0.5% PBS/Tween 20, exposed to anti-EMAP-II antibodies R2B2, and diluted in 0.5% PBS/Tween 20 for 2 h at room temperature. Proteins were visualized with HRP-conjugated goat anti-rabbit immunoglobulin G (Sigma) using the enhanced chemiluminescence system (Amersham).
Assessment of apoptosis by fluorescein isothiocyanate (FITC)-labeled Annexin-V and propidium iodide (PI)
Annexin-V binds to phosphatidylserine exposed on the external surface of the plasma membrane during the early phase of the apoptotic program, and DNA within the nucleus only becomes accessible to PI during late apoptosis. Therefore, these two reagents can be used together to distinguish early and late apoptosis. Peripheral blood mononuclear cells (PBMC) and Jurkat cells, following treatment with rEMAP-II or tumor cell-conditioned medium or after coculture with tumor cell monolayers, were assessed for apoptosis. Untreated control cells and cells treated with 200 ng/ml TNF-/IFN- were also prepared. Samples were immediately analyzed using the Apotest-FITCTM kit (Dako, Denmark) in accordance with the manufacturer’s instructions by flow cytometry. Analysis of PBMC was restricted to the lymphocyte population by gating on forward- and right-angle scatter. Twenty thousand events were acquired for each analysis.
Conditioned medium experiments
DLD-1 cells were grown for 48 h in serum-free RPMI medium or in medium supplemented with TNF-/IFN- (200 ng/ml each). Used culture medium was centrifuged at 2000 g for 5 min to remove cellular debris and stored at –80°C. Jurkat cells were pelleted, resuspended in conditioned medium, and cultured for 24 h. The cells were then centrifuged at 1000 g for 5 min, resuspended in PBS, and analyzed for apoptosis as described above. In some experiments, R2B2 antibodies against EMAP-II (final concentration, 10 µg/ml) were added to the incubation mixture of Jurkats and conditioned medium to block endogenous EMAP-II. We have previously demonstrated functional blocking with this concentration of R2B2 antibodies [5
].
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RESULTS
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Human cytokine EMAP-II is identical to the p43 auxiliary protein
By database mining, we identified a 117-kbp genomic contig, located in the human chromosome 4q22–25 region, which corresponds to the human EMAP-II gene. Within this contig, we have identified coding regions identical in sequence to the human EMAP-II cDNA (accession no. U10117). The human EMAP-II gene is 30 kbp in size, comprising seven exons (seeFig. 1
). It appears to be arranged in head-to-head configuration with another as-yet uncharacterized gene known in the expressed sequence tag database as HPSC302. We have cloned a 360-bp fragment incorporating the intergenic region and shown that this sequence has strong promoter activity in 293 cells in either orientation (data not shown).
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Figure 1. Structure of the human EMAP-II gene, located in chromosome region 4q22–25. The EMAP-II gene is contained within seven exons and spans 30 kb. EMAP-II is transcribed in the opposite direction to a novel gene HSPC302 and shares an intergenic region of approximately 290 bp. Exons are indicated by short, vertical lines, and exon size is indicated underneath in bp. Intron size is indicated vertically in kb.
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By 5' RACE, we demonstrated that the transcription of EMAP-II was found to initiate at three positions, all within 15 nucleotides of each other, to give a first exon of approximately 86 nucleotides. These three starts of transcription are all located no more than 63 nucleotides upstream of the published EMAP-II sequence of Kao et al. [4
] (accession no. U10117). This additional 5' sequence shows sequence divergence with the hamster p43 sequence. As a consequence, the hamster sequence contains a translation start site appropriate for a predicted p43 protein as well as a 34-kD EMAP-II protein, and the human sequence contains a start site for only a 34-kD protein but not p43 (Fig. 2A
).
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Figure 2. (A) Potential translation start sites (ATG codons) within human and hamster p43/EMAP-II transcripts. Note that the human gene is likely to give rise to only one 34-kDa protein. (B) Western blot with polyclonal antibodies against human EMAP-II, showing two proteins arising in Chinese hamster ovary (CHO) cells (Ha) at the predicted 43 kDa and at 34 kDa but only one in human endothelial cells (Hu), corresponding to 34 kDa EMAP-II. ATG.
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These sequence data are in agreement with the results of Western blots with our polyclonal antibodies against EMAP-II, which reveal the presence of 43 kDa and 34 kDa bands in extracts from hamster (CHO) cells (Fig. 2B)
. Human cell extracts, however, only demonstrate a 34-kDa band. Thus, it appears that humans do not express a homologue of the hamster p43 protein. The reasons for this evolutionary divergence are currently unclear.
We have found that a wide range of cultured cells, normal and transformed, retain the 34-kDa precursor form of EMAP-II in the cytoplasm. In some tumor cell lines, however, fully or partially processed EMAP-II can be detected in the culture medium [5
, 9
, 16
]. We have previously shown that in most normal tissues and in cell lines, the fully processed form migrates with a mobility between 18 and 20 kDa, although the recombinant form that we have used, which contains some extra amino acid sequence, moves with mobility nearer 22 kDa [5
, 9
, 16
]. Figure 3
shows a Western blot of a cell extract and highly concentrated conditioned medium from DLD-1 colorectal carcinoma cells. DLD-1 cells contain the 34-kDa form but release low levels of a fully processed, 18–20 kDa form of EMAP-II.
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Figure 3. Western blot of EMAP-II in extract and cell-culture supernatant from the DLD-1 human colorectal carcinoma cell line. Lane 1, rhEMAP-II (22-kDa form); lane 2, DLD-1 cell extract; lane 3, concentrated DLD-1 cell supernatant.
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The extracellular form of EMAP-II induces apoptosis in activated lymphocytes
The effect of EMAP-II on lymphocytes has not previously been described. We performed a series of experiments to examine the effects of EMAP-II on PBMC and Jurkat T cells (a model for activated T cells). PBMC were treated with rEMAP-II before stimulation with phytogemagglutinin to induce lymphocyte proliferation. EMAP-II caused a dose-dependent inhibition of [3H]-thymidine incorporation (50% suppression at 40 nM) but had no effect on unstimulated PBMC [17
].
We then sought to determine whether this effect was cytostatic or cytotoxic and assessed levels of apoptosis in Jurkat T cells and activated PBMC following exposure to recombinant or tumor cell-derived EMAP-II. Figure 4
shows the results, pooled from three experiments, of measuring apoptosis in Jurkat cells at 4 and 24 h after incubation with 100 nM rEMAP-II, which also induced apoptosis in mitogen-treated PBMC but not in unstimulated PBMC [17
].
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Figure 4. Effect of rEMAP on Jurkat T cells. Bar graph shows percentage of apoptotic Jurkat cells, as measured by the Annexin V/PI method at 4 and 24 h after treatment with rEMAP-II (100 nM). Solid bars, Untreated cells; hatched bars, cells treated with EMAP-II. Data represent mean of three determinations ± 1 SD. At 4 h and 24 h, there was a significant difference between control and EMAP-II-treated cells. P < 0.05.
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We also examined the effects of growth medium conditioned by DLD-1 colorectal adenocarcinoma cells on Jurkat cells to determine whether native EMAP-II protein produced by the tumor cells induces cell death in lymphocytes. DLD-1 cells express the 34-kDa form of EMAP-II intracellularly; however, they secrete significant quantities (2–10 ng/ml/24 h) of the 20- to 22-kDa form of EMAP-II into the culture medium in response to the combination of TNF- and IFN- [17
]. When Jurkat cells were exposed to medium conditioned by untreated DLD-1 cells, there was no effect on viability (Fig. 5a
). The addition of fresh combination TNF-/IFN- to this medium induced some early signs of apoptosis in Jurkat cells (b), indicating that the cytokine combination alone induces some apoptosis. However, conditioned medium from DLD-1 cells pretreated with TNF-/IFN- for 48 h induced a further 4- to 5-fold increase in apoptosis (in Jurkat cells) (c). Critically, this effect was partially reversed by addition of polyclonal antibodies against EMAP-II to the incubation mixture (d).
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DISCUSSION
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These data suggest that EMAP-II released by tumor cells is cytotoxic to activated lymphocytes and therefore, may constitute a component of a novel, immunosuppressive pathway in solid tumors. Further experiments [17
] have indicated that in some colorectal tumor cell lines (e.g., HT29), EMAP-II is not released in soluble form but is expressed on the cell surface. Coculture of these cells with Jurkat cells leads to Jurkat apoptosis, which is partially inhibited by anti-EMAP-II antibodies [17
].
We do not understand the mechanism by which the 34-kDa intracellular form of the p43/EMAP-II form is processed to smaller forms and subsequently found in the supernatants of cells such as DLD-1. The 34-kDa form lacks a hydrophobic signal peptide necessary for membrane translocation nor is it predicted to be glycosylphosphatidylinositol-anchored. There is also conflicting evidence concerning the susceptibility of EMAP-II to proteolytic cleavage: One study [8
] suggests pro-EMAP-II is cleaved at a critical aspartate residue by caspase 7, but other, more recent studies [9
, 10
] could not confirm this.
EMAP-II triggers apoptosis in activated lymphocytes, but the mechanism by which it does so is not known. It could act directly through death receptors, grouping it with TNF, FasL, and TNF-related apoptosis-inducing ligand or indirectly, inducing or potentiating one of these death ligand/receptor interactions. Furthermore, it appears that EMAP-II can induce apoptosis through direct cell–cell contact or by acting as a soluble mediator. This pathway is restricted to a limited number of target cells, including endothelial cells and activated lymphocytes. Elucidation of the receptor(s) for EMAP-II is clearly an important challenge lying ahead. Another important question relates to why the p43 form of the protein makes its way to the cell surface of tumor cells to take on the EMAP-II role.
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ACKNOWLEDGEMENTS
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A Cancer Research UK Program Grant to J. C. M. supported this work.
Received October 19, 2003;
revised January 12, 2004;
accepted January 22, 2004.
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