Difference in cytokine production and cell cycle progression induced by Epstein-Barr virus Lmp1 deletion variants in Kmh2, a Hodgkin lymphoma cell line
© Sueur et al.; licensee BioMed Central Ltd. 2014
Received: 15 January 2014
Accepted: 8 May 2014
Published: 19 May 2014
Epstein-Barr virus (EBV) is associated with 20-40% of Hodgkin’s Lymphoma (HL) cases. EBV-encoded latent membrane protein 1 (LMP1) is a well-known oncogenic protein and two C-terminal deletion variants, del30-LMP1 and del69-LMP1, have been described in animal models to be more tumorigenic than the wild-type form. This work aims to detail the implication of LMP1 in the development of HL and to characterize the particular effects of these variants.
We established HL-derived cell lines stably transfected with the pRT-LMP1 vector coding for the EBNA1 gene and allowing expression of the different LMP1 variants under the control of a doxycyclin-inducible promoter. Communication between cells was assessed by measuring the expression of various pro-inflammatory cytokines by flow cytometry after intracellular LMP1 and cytokine double staining. Proliferative properties of LMP1 variants were also compared by studying the repartition of cells in the different phases of the cell cycle after EdU incorporation combined to LMP1 and DAPI staining.
All LMP1 proteins induced the expression of several pro-inflammatory cytokines such as TNF-α, TNF-β, IL-6, RANTES/CCL5 and IFN-γ. However, the del30-LMP1 variant induced cytokine expression at a lower level than the other variants, especially IFN-γ, while the del69-LMP1 variant stimulated greater cytokine expression. In addition, we measured that all LMP1 proteins greatly impacted the cell cycle progression, triggering a reduction in the number of cells in S-phase and an accumulation of cells in the G2/M phase compared to the HL-non induced cells. Interestingly, the del30-LMP1 variant reduced the number of cells in S-phase in a significantly greater manner and also increased the number of cells in the G0/G1 phase of the cell cycle.
Weak IFN-γ expression and specific alteration of the cell cycle might be a way for del30-LMP1 infected cells to escape the immune anti-viral response and to promote the development of cancer. The differences observed between the LMP1 variants reflect their own oncogenic properties and eventually impact the development of HL.
EBV is an ubiquitous tumor-causing virus infecting more than 90% of human adults worldwide with a life-long infection in B lymphocytes asymptomatically . EBV infection in HRS cells presents a type II latency program, characterized by the expression of the latent membrane protein 1 (LMP1) and two other viral proteins, EBNA1 and LMP2 in addition to small RNAs, termed EBER 1 and 2 . LMP1 is a multifunctional oncoprotein essential for EBV-induced B-cell proliferation and transformation in vitro, as it shares several features with CD40, a member of the tumor necrosis factor receptor family (TNFR). LMP1 activates the transcription factor nuclear factor-κB (NF-κB) by promoting turnover of IκBα, an important inhibitor of NF-κB, conferring the cells a protection against apoptosis. A direct link between LMP1 and cell cycle progression has also been shown in several studies although they were essentially focused on NPC cells or Burkitt lymphoma cell lines [4–7].
In the EBV-associated pathologies, different variants of LMP1 have been described. For example, a 30-bp deletion variant at the C terminal and corresponding to the CAO variant isolated from a NPC patient, was found to have increased potential to transform rodent fibroblasts and to induce tumors in nude mice when compared to wild-type LMP1 . A 69-bp deletion LMP1 variant has also been described in NPC  and other lymphoproliferative disorders such as HL . These deletions do not interfere with the stimulation of NF-κB . However, the presence of such variants associated to the pathogenesis of several diseases leads to the hypothesis that polymorphisms within LMP1 gene might influence the susceptibility to develop EBV-associated tumors.
Hodgkin Lymphoma (HL) is a B cell lymphoma, characterized by a minority of neoplastic cells, the Hodgkin and Reed-Sternberg cells (HRS cells), and an extensive inflammatory background. Classical HRS cells derive from post-germinal center B cells, destined for apoptosis in the B cell selection process because of the lack of successful immunoglobulin gene rearrangement. In Western world, 20-40% of HL are associated with Epstein-Barr Virus (EBV) while in developing countries it reaches 70% . Interestingly, EBV has been detected in 80-100% of HL arising in HIV patients, supporting the notion that this virus plays a pivotal role in the pathogenesis of this tumor . Moreover, presence of the 30-bp deletion in LMP1 has been frequently associated with the HL disease in HIV infected individuals [13, 14]. Recently, LMP1 has been shown to increase the DDR1 tyrosine kinase receptor in germinal center B cells, activating the expression of pro-inflammatory mediators and protecting lymphoma cells from cell death . Besides, many studies have documented that HL is associated with disturbed cytokine production . Cytokine and chemokine production may not only promote growth of HRS cells and help to evade immune surveillance, but they also cause the characteristic histology and the clinical symptoms of HL . Cross-talk between HRS cells and surrounding lymphocytes has been studied for many years, and this interaction appeared to play an important role for the pathogenesis of HL . Few studies have documented the impact of EBV infection on HL development. HL frozen tissues  or derived cell lines infected by EBV in vitro or transiently transfected by a constitutive expressed LMP1 vector were used [20–24]. However, results obtained from these studies were difficult to interpret since either there were not quantitative or the cell lines did not express LMP1 until a membrane signal was applied (CD40 ligand and IL4), leading to morphological studies where LMP1 was linked to the formation of multinuclear cells or showing differentially expressed proteins by microarray RNA assays, not confirmed by protein expression techniques. Other studies about LMP1 genetic diversity from samples derived from HL patients focusing mainly on LMP1 variant origin and activation of the NF-κB pathway were also conducted [25–27]. However, the impact of the LMP1 polymorphism on the HL cells has not been documented.
In this study, we investigated whether WT-LMP1 and the deletion variants del30-LMP1 and del69-LMP1 could modulate cytokine expression and cell cycle progression in KMH2 – a HL derived cell line – to analyze the impact of LMP1 polymorphism on the development of HL.
Characterization of the KMH2-pRT-LMP1 established cell lines
LMP1 induces expression of several cytokines in KMH2 cells
Cytokine expression induced in Hodgkin’s lymphoma-derived cells
del30-LMP1 induces cytokine expression at a lower level than the other LMP1 variants
To sum up, del30-LMP1 tended to induce cytokine expression at a lower level compared with both forms: WT-LMP1 and del69-LMP1. Nonetheless, del69-LMP1 seemed to induce more cytokine expression than the two other LMP1 variants.
LMP1 variants influence differently cell cycle progression
Thus, all variants reduced the S phase and accumulated cells in the G2/M phase, suggesting a blockade at the G1/S checkpoint and a block to completion of mitosis in the KMH2 cells (Figure 3e). The del30-LMP1 variant significantly reduced the number of S-phase cells and further increased the G0/G1-phase cells.
In the context of HL, where tumor cells represent only 0.1 to 1% of the cells surrounded by fibroblasts and immune cells, communication between cells mediated by cytokines and chemokines is of upmost importance. LMP1 is a well-known oncogene involved in many cellular signaling pathways and thus able to interfere in cytokine secretion. Previously, LMP1 has been shown to induce IL-6 and IL-8 secretion in epithelial cells  via the NF-κB pathway  and IL-10 in Burkitt lymphoma cells . In LCL, LMP1 triggers cell proliferation by inducing cytokines such as CCL3 and CCL4 through the JUN-kinase pathway , and also uses the NF-κB pathway to stimulate IFN secretion . In this study, we showed that LMP1 induced significantly the expression of several cytokines in LMP1 positive KMH2 HL-derived cells, such as IFN-γ, IL-6, RANTES/CCL5, TNF-β and TNF-α. These cytokines are mainly pro-inflammatory, attracting immune cells in the microenvironment and known to be implicated in the development of HL [17, 34]. Vockerodt et al.  showed that LMP1 induced high levels of IL1, IL8 and RANTES mRNAs in primary human tonsillar germinal center B cells (CD10+ cells). We did not find IL8 and IL1 elevated amounts in the LMP1 positive KMH2 cell lines. Whether this discrepancy is due to the different techniques used or to the cells still remains to be determined.
Little is known about the influence of LMP1 on cytokine production in the particular context of HL. An histological study showed that IL-6 was more often expressed in LMP1-positive HRS cells compared to the negative ones . Showing differences in cytokine expression between the three LMP1 positive forms could be of interest to HL. While a significantly higher number of del69-LMP1 cells expressed IL-6 and RANTES, del30-LMP1 cells expressed IFN-γ, IL-6 and TNF-β in a significantly lesser extent than the other variants. IFN-γ is involved in the anti-viral response against EBV-infected cells. Weak IFN-γ expression might be a way for del30-LMP1 infected cells to escape the immune anti-viral response. Besides, on the one hand, the presence of cytokines is necessary for cell survival and tumor promotion. Yet, on the other hand, an excessive cytokine secretion stimulates the recognition and degradation of EBV-infected cells by the immune system. The low level of cytokines expressed in del30-LMP1 KMH2 cells could be a mechanism for EBV to promote the development of HL while escaping the immune surveillance. These observations are consistent with the high frequency of del30-LMP1 variant observed among HIV positive people developing a HL [13, 14]. On the contrary, the high proportion of del69-LMP1 HL cells to express pro-inflammatory cytokines could trigger their recognition by the immune system and would explain the low frequency of the del69-LMP1 variant in vivo, described only sporadically in NPC or some lymphoproliferative diseases. Prognostic significance of pretreatment serum cytokines in HL has been described . In particular, they show that serum levels of IL6 and IL2R may be used to identify patients with HL at risk for early-disease relapse. Since we found that IL6 was less induced by the del30-LMP1 variant, it thus would be of great interest to determine in vivo, if the IL6 level in serum may be linked to the presence of del30-LMP1 variant and of early-disease relapse.
Oncogenic LMP1 has also a great influence on cell proliferation and survival by contributing to cell cycle progression. Here we show for the first time in HL cells, that all LMP1 variants interfered with cell cycle progression. LMP1 caused a decrease in the S-phase cell population, particularly significant with the del30-LMP1 variant and an increase of the G2/M-phase cells. The del30-LMP1 variant provoked an additional accumulation of cells in the G0/G1 phase. These observations were consistent with previous studies performed in NPC cell lines and LCLs with WT-LMP1 or a NPC-derived variant, carrying the 30 bp deletion [4, 7]. LMP1 has been described to impair G2 checkpoint leading to a possible accumulation of cells with unrepaired DNA damages  or to promote cell cycle progression by accelerating the G1/S transition [5, 37]. On the other hand, LMP1 has also been described to have cytostatic or even cytotoxic effects when expressed at high levels [7, 38]. A recent work suggests that the ability of LMP1 to exert both cytotoxic and pro-proliferative properties is necessary during the transformation process . While LMP1 induces cell death in the microenvironment, only EBV infected cells expressing the oncogenic LMP1 survive . The fact that LMP1 variants do not elicit the same effect on cell cycle progression suggests that they have different impact on cell cycle checkpoints. This could directly reflect on their oncogenic potential. Besides, cytokines themselves can influence the cell cycle progression. For example, IL-8 is often overexpressed in cancers and contributes to the tumor development by promoting the G1/S transition of cell cycle . It would be interesting to further investigate the cellular mechanism involved in cell cycle progression with each LMP1 variant and particularly the impact of the LMP1-induced cytokines.
In conclusion, this study brings new insights into the impact of LMP1 on cytokine expression and cell cycle progression in HL. We highlight differences between LMP1 variants which could partly be responsible for their respective oncogenic properties and explain their implication as risk factors in the development of HL.
Materials and methods
We performed an overlapping PCR using the p2167 plasmid (kindly provided by W. Hammerschidt, Munich, Germany), in order to create the 30-bp and 69-bp deletions in the BNLF1 gene coding for LMP1 . The p2167 plasmid contains 9.5 kb of the EBV genome, as described . Two DNA fragments surrounding the deletions (respectively in position 1190-1229 and 1161-1229) were amplified by PCR with a high fidelity polymerase (Pfu, Promega, Fitschburg, WI, USA). The following primers were used,
First, two PCRs were performed with 50 ng of the p2167 plasmid in a total volume of 50 μl, with primers P1 + P2a and P3a + P4 for the 30-bp deletion or P1 + P2b and P3b + P4 for the 69-bp deletion. After 5 min of denaturation at 95°C, 10 cycles of 30 sec at 95°C, 30 sec at 55°C and 2 min at 72°C were carried out, followed by an elongation step for 5 min at 72°C. PCR products were purified by QIAEX purification kit (Qiagen, Hilden, Germany), quantified by the nanodrop system (Labtech, Uckfield, United-Kingdom) and equimolarly mixed to be submitted to a second PCR. After 10 cycles with the same program but without primers, allowing the first two PCR products to hybridize, external primers P1 and P4 were added for another 20 cycles. The final 1.6 kb PCR product containing the 30-bp or 69-bp deletion was reintroduced into the original p2167 plasmid in the NheI and Bst1107I restriction sites and subcloned into the Sfi-I sites of the pRT-LMP1 vector (kindly provided by J. Feuillard, Limoges, France) with the In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA). This vector includes the EBNA1 and hygromycin B resistance genes, that favour maintenance of the vector as an episome in the cells, and allow selection of transfected cells respectively. A bidirectional tetracyclin-inducible promoter drives the expression of LMP1 and NGFRt . The pcDNA-WT-LMP1 plasmid is a kind gift from F. Grässer (Hambourg, Germany).
Cell culture, transfection and induction
293-HEK cells (human embryonic kidney cell line) were cultured in DMEM supplemented with heat inactivated fetal bovine serum 10% and antibiotics (100 units/ml penicillin/streptomycin and 1 μg/ml ofloxacin). KMH2 cells (EBV-negative Hodgkin Lymphoma-derived cell line obtained from DSMZ, Braunschweig, Germany) were cultured in RPMI supplemented with 10% heat inactivated fetal bovine serum and antibiotics (100 units/ml penicillin/streptomycin, 1 μg/ml ofloxacin), at 37°C in a 5% CO2 atmosphere. For transient transfection of pcDNA-WT-LMP1 in 293-HEK cells, 5.105 cells were plated in a 35-mm petri dish and transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in the ratio of 10 μl transfection reagent for 4 μg of DNA, following the manufacturer’s instructions. Establishment of KMH2 cell lines stably transfected with the three pRT-LMP1 plasmids (pRT-WT-LMP1, pRT-del30-LMP1 and pRT-del69-LMP1) was performed with the Amaxa electroporation system (Amaxa, Cologne, Germany), according to the manufacturer’s instructions. Five millions KMH2 cells were electroporated with 5 μg of DNA, using the Nucleofactor kit-T (Amaxa) and the T-001 program. Cells were then resuspended in 5 ml of fresh RPMI with 20% FBS, supplemented with 1X non-essential amino-acids, 10 mM Hepes and 1 mM sodium pyruvate. Hygromycin was introduced in the medium 48 h after the transfection at 200 μg/ml. After three weeks of selection, LMP1 expression was assessed by flow cytometry after inducing the cells with 1 μg/ml doxycyclin (Clontech) for 24 h, as described elsewhere .
Five millions KMH2-pRT-LMP1 cells were grown in presence or not of doxycyclin for 24 h. Cells were lysed in a buffer composed of 50 mM Tris pH 6.8, 2% SDS, 10% glycerol and protease inhibitors. Lysates were sonicated and total proteins were quantified with BCA kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Fifty micrograms of total proteins were separated on a 9% SDS-PAGE gel, transferred on a PVDF membrane and revealed by western-blot analysis with an anti-LMP1 antibody (monoclonal CS.1-4, Dako, Glostrup, Denmark, 1:100) and a rabbit polyclonal anti-actin (Sigma, St Louis, MO, USA; 1:400). Secondary HRP-conjugated anti-mouse (1:20000), anti-rabbit antibodies (1:10000) were purchased from Invitrogen). The signal was developed by Supersignal West Pico Chemiluminescent Substrate (Pierce). The intensity of protein bands was quantified with Adobe Photophop CS3 software.
RNA extraction and RT-PCR
Total RNA was extracted from 5.106 KMH2-pRT-LMP1 cells with TRI-Reagent (Euromedex, Souffelweyersheim, France), quantified with a nanodrop ND1000 instrument system (Labtech) and treated with Turbo-DNase (Ambion, Austin, TX, USA). cDNA was synthesized from 1 μg total RNA with random hexamers, using the superscript III (Invitrogen) in a total volume of 20 μl. PCRs were then performed on 1 μl of cDNA, with 0.25U of the Phusion polymerase (Thermo-Scientific, Waltham, MA, USA) in the provided buffer, in presence of 200 μM dNTP and 200 μM of specific primers. Primer sequences are: LMP1-forward, 5′-CCTCATAGCCCTAGCGACTC-3′ ; LMP1-reverse, 5′-GTCGTCATCATCTCCACCGG-3′ ; EBNA1-forward, 5′-CCGCAGATGACCCAGGAGAA-3′ ; EBNA1-reverse, 5′-TGGAAACCAGGGAGGCAAAT-3′ ; β-actin-forward, 5′-CGTGATGGTGGGCATGGG-3′ β-actin-reverse, 5′-CTGGGTCATCTTCTCGCG-3′. After a denaturation step of 3 min at 94°C, the following cycle was applied to the samples : 30 sec at 94°C, 30 sec at 60°C (for EBNA1, TH was 56°C), 30 sec at 72°C, repeated 26 times for the actin amplification and 30 times for LMP1 and EBNA1 amplifications. A final elongation step was performed for 5 min at 72°C. PCR products were migrated on a 2% agarose gel in 1X TAE buffer and visualized with ethidium bromide staining (Invitrogen).
LMP1 staining and intracellular cytokine detection
KMH2 cells were either treated by doxycyclin (1 μg/ml) to induce LMP1 expression or stimulated with 50 ng/ml PMA (Sigma) and 1 μg/ml ionomycin (Sigma) to be used as positive control for cytokine expression during 24 h. Throughout the last 4 h of doxycyclin or PMA-ionomycin induction, cells were treated with 10 μg/ml brefeldin A (BioLegend, San Diego, CA, USA) and 2 μM monensin (Sigma) to block secretion of cytokines. Cells were collected and fixed with fixation buffer (BioLegend) for 20 min at room temperature. Cells were then permeabilized by two centrifugations (for 8 min at 350 g) with permeabilization wash buffer (BioLegend) allowing sequential double immunolabeling of LMP1 and cytokines of interest. LMP1 staining was performed primarily as follows: cells were saturated for 30 min in 100 μl RPMI containing 10% human plasma (kindly provided by EFS Rhône-Alpes, Grenoble, France) and 0.1% triton X-100, incubated for 30 min with the anti-LMP1 antibody (1:100) and washed twice with 5 ml of RPMI. Cells were then incubated for 30 min with an Alexa A488 or A594-conjugated anti-mouse antibody (Invitrogen, 1:2500) and washed twice with 5 ml of RPMI. An anti-mouse secondary antibody free Fab sites was finally satured with a mouse hybridoma supernatant (clone D3210, kindly provided by E. Drouet, UVHCI, Grenoble, France). In a second step, intracellular cytokine staining was carried out by resuspending cells in 100 μl of permeabilization wash buffer supplemented with the anti-cytokine antibody and incubated for 20 min at room temperature, protected from light. Cells were then washed twice with 2 ml of permeabilization wash buffer and finally resuspended in cell staining buffer (BioLegend) for flow cytometry analysis on a MACSQuant VYB (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany). Anti-cytokine antibodies used were APC-conjugated anti-human TNF-α (clone Mab11) or its mouse isotype control (clone MOPC21), PE-conjugated anti-human IL-6 (clone MQ2-13A5), anti-human IL-8 (E8N1), anti-human TNF-β (clone 359-81-11), anti-human TGF-β (TW4-2 F8), anti-human IL-1α (364-3B3-14), rat isotype control (clone RTK2071) and mouse isotype control (clone MOPC21) from BioLegend, PE-conjugated anti-human IFN-γ (clone 4S.B3), anti-human IL-9 (MH9A3) and anti-human RANTES/CCL5 (clone 2D5) from BD Pharmingen (San Diego, CA, USA), or FITC-conjugated anti-human IL1-RA (CRM17) and its mouse isotype control (P18.104.22.168.1) from eBioscience (San Diego, CA, USA).
Cell cycle and proliferation
In order to determine the percentage of cells in the different phases of cell cycle, we used the EdU-click 555 (Jena Bioscience, Jena, Germany) combined with LMP1 and DAPI staining. EdU is a nucleoside analogue to thymidine incorporated into DNA, it reveals the proliferating cells in S phase. DAPI measures the total quantity of DNA in the cells and the LMP1 labelling enables to select only the LMP1-expressing cells for our study. Cells were induced with doxycyclin for 24 h and, during the last 150 min, 10 μM EdU was added in the culture medium. Cells were, then, fixed with fixation buffer and permeabilized with permeabilization wash buffer (BioLegend). For EdU revelation, cells were resuspended in the cocktail-click containing TAMRA-azide, incubated for 30 min at room temperature protected from light and washed twice with 6 ml PBS. LMP1 staining was performed as described previously for intracellular cytokine detection and cells were finally incubated in PBS with 1 g/L glucose, DAPI 1:1000 and 100 μg/ml RNAseA until flow cytometry analysis.
Means, standard deviations and p-values were calculated with the GraphPad InStat3 software (San Diego, CA, USA). All bar graphs represent means as per at least three independent experiments for cytokine detection and at least five independent experiments for cell cycle studies. Error bars represent standard deviations. To assess statistical differences, means were compared through a one-way ANOVA test followed by a Tukey post-test for multiple comparison significance test between the analyzed groups. When specified, for single comparison of a population with the control cell group, the ANOVA test was followed by a Dunnett post-test. * stands for p < 0.05, ** for p < 0.01 and *** for p < 0.001.
This research was supported by the Agence Nationale de Recherches sur le Sida et les hépatites virales (ANRS-Lymphovir sub-study), Sidaction, the Centre National de Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the University Joseph Fourier, the Grenoble University Hospital. We thank Evelyne Manet, Henri Gruffat (ENS-Lyon, France) and Xavier Ronot (FRE AGIM CNRS-UJF 3405, Grenoble, France) for helpful advice and discussions. We thank Rose-Laure Revel-Goyet, Françoise Lacroix and Jean-Philippe Kleman (Institut de Biologie Structurale, Grenoble) for the support and access to the Cell imaging Platform. This work used the platforms of the Grenoble Instruct center (ISBG ; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). V. B. is an INSERM scientist.
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