1 Laboratoire de Virologie Moléculaire et Structurale, UPRES-EA2939, GDR CNRS 2372, Université Joseph-Fourier Grenoble I, 38706 La Tronche, France
2 U412 INSERM, ENS-Lyon, 46 allée d'Italie, 69364 Lyon Cedex 07, France
Correspondence
Alain Sergeant
alain.sergeant{at}ens-lyon.fr
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ABSTRACT |
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INTRODUCTION |
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hIL-10 has been described as a potent immunosuppressive cytokine since it affects cell-mediated immune responses. hIL-10 is otherwise a potent growth and differentiation factor for B cells and also protects some B cells from apoptosis. In contrast to normal non-activated B cells, EBV-positive B cells produce hIL-10, which stimulates cell growth (Moore et al., 2001). Several observations link hIL-10 production and EBV infection. Analysis of nasopharyngeal carcinoma specimens showed that the number of cytotoxic T cells was significantly lower in hIL-10-positive than in hIL-10-negative tumours (Yao et al., 1997
). hIL-10 abrogates the EBV-specific memory response defined as the ability of T cells to inhibit the growth of EBV-infected B-lymphocytes (Bejarano & Masucci, 1998
). In HD cases, upregulation of hIL-10 expression was linked to detection of the EBV genome in tumour cells and was associated with a low number of cytotoxic T lymphocytes surrounding the tumour (Herbst et al., 1996
; Ohshima et al., 1995
). Finally, a significant increase in the circulating hIL-10 level has been observed in vivo before EBV-associated post-transplant lymphomas and non-Hodgkin's lymphoma diagnosis (Birkeland et al., 1999
; Cortes & Kurzrock, 1997
). All these studies suggest that hIL-10 may play a role in EBV-related neoplastic diseases.
EBV-associated induction of hIL-10 production occurs at the transcriptional level. The viral gene products that induce hIL-10 expression have been characterized. The LMP-1 protein and the ubiquitously expressed EBV-encoded small non-polyadenylated RNAs, EBER1 and EBER2, both induce hIL-10 expression, through the p38/SAPK2 pathway and in a PKR-independent way respectively (Kitagawa et al., 2000; Nakagomi et al., 1994
; Vockerodt et al., 2001
). In addition, hIL-10 expression is induced on activation of the EBV lytic cycle in an EBV-carrying B-cell line by mechanisms that have still to be defined (Sairenji et al., 1998
).
The productive cycle is initiated by the expression of a viral transcription factor, originally named EB1 (Chevallier-Greco et al., 1986), and later called Zta, BZLF1 or ZEBRA (Countryman et al., 1987
; Lieberman et al., 1990
). EB1 is a bZIP-like protein (basic Zipper) that displays sequence homology with proteins of the AP-1 family (Farrell et al., 1989
; Urier et al., 1989
). EB1 activates the transcription of viral genes (Kieff & Rickinson, 2001
) and Orilyt-dependent viral DNA replication (Schepers et al., 1993
) through binding to specific DNA sequences called Z-Responsive Elements (ZRE) (for references see Speck et al., 1997
). EB1 also activates the transcription of cellular genes such as c-fos (Flemington & Speck, 1990
), and those encoding TGF-
(Cayrol & Flemington, 1995
), TKT (Lu et al., 2000
) and MMP-9 (Yoshizaki et al., 1999
).
In this study, we show that EB1 activates endogenous hIL-10 transcription and secretion in an EBV-negative B cell line. We further demonstrate in transient transfection assays that the viral protein activates transcription at the hIL-10 promoter, linked to the luciferase reporter gene. We have localized EB1-binding sites in the hIL-10 minimal promoter by DMS interference. Mutation of these sites confirmed that EB1 directly activates transcription at the hIL-10 promoter.
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METHODS |
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The EB1 and EB1 mutant expression vectors have been described elsewhere (Manet et al., 1989; Giot et al., 1991
). Briefly, plasmid pSG-Z41 contains a cDNA coding for EB1 placed under the control of the SV40 early promoter-enhancer. Alanine at position 185 was replaced by a lysine residue in the EB1 mutant Z311. Amino acids between positions 25 and 42 were deleted in the EB1 mutant
25-42 (see Fig. 3A
).
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Detection of secreted hIL-10 by ELISA.
1x107 cells (DG75) were transfected as described above with the EB1 expression vector pSG-Z41, and resuspended in a final volume of 5 ml of complete medium. At different times following transfection, 200 µl of the culture medium was collected. The hIL-10 content of the supernatants was measured by ELISA as described elsewhere (Badou et al., 2000). The anti-hIL-10 monoclonal antibody MAB217 (R&D Systems) was used as the coating antibody and the biotinylated anti-hIL-10 polyclonal antibody BAF217 (R&D Systems) was used as the detection antibody. Absorbance was read at 490 nm. hIL-10 production was quantified from a standard curve generated by using various concentrations of recombinant human hIL-10 (R&D Systems). In our assay, the limit of detection of hIL-10 was about 3050 pg ml-1.
Western blot analysis.
Cells were collected after transfection, washed with cold PBS and incubated with 100 µl of lysis buffer (0·05 M Tris/HCl, 10 % glycerol, 2 % SDS, 5 % -mercaptoethanol, 0·05 % bromophenol blue). Equal amounts of protein were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham). The membrane was then incubated with the EB1 monoclonal antibody Z125 (Mikaelian et al., 1993
) and further incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulins (Amersham). The proteins were visualized with the ECL kit (Amersham).
Electrophoretic mobility shift assay (EMSA).
The DNA probes used were either obtained by DdeI digestion of the (-371/+33) hIL-10 promoter fragment (see Fig. 4A) or were synthetic oligonucleotides of length 20 bp (see Fig. 5A
). Double-stranded oligonucleotides were 5'-end labelled with [
-32P]ATP (Amersham) using polynucleotide kinase (Invitrogen). Labelled probes were purified by electrophoresis in a 6 % polyacrylamide gel. EB1-His-tagged was purified using Ni2+NTA agarose (Qiagen) according to the manufacturer's recommendations. 2 µl of EB1-His-tagged was incubated with 2x104 c.p.m. of each labelled oligonucleotide. Incubations were for 20 min at 4 °C, in a volume of 20 µl containing 20 mM Tris-HCl (pH 7·9), 1 mM MgCl2, 0·5 mM DTT, 15 % glycerol, 100 mM KCl, 0·2 µg poly(dI : dC) and 5 µg BSA. The EB1DNA complexes were separated from the non-complexed DNA by gel electrophoresis and were visualized by autoradiography as previously described (Giot et al., 1991
). In competition experiments, the non-labelled double-stranded oligonucleotide ZRE, carrying two EB1-binding sites (5'-TATGCATGAGCCACAGGCATTGCTAATGTA-3') was used as a specific competitor at a 100-fold molar excess (the EB1-binding sites are underlined).
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RT-PCR
Total RNA.
This was extracted from 1x107 cells and purified by CsCl centrifugation as described (Shaw et al., 1985). Purified RNA was resuspended in RNase-free water.
First-strand cDNA synthesis.
RNA was denatured for 10 min at 70 °C and then chilled on ice. First-strand cDNA synthesis was performed from 5 µg of total RNA at 42 °C for 50 min in a final volume of 20 µl: 5 µl of denatured RNA, 4·5 µl of 5x buffer, 2 µl 0·1 M DTT, 1 µl 10 mM dNTP mix (Invitrogen), 1 µl of 1 µg oligo(dT) (Biolabs) ml-1, 1 µl of Superscript II (200 units µl-1) (Invitrogen). Tubes were then heated for 15 min at 70 °C.
Cytokine cDNA amplification.
PCR mixture (23 µl) was added to 2 µl of first-strand cDNA. PCR mixture contained 2·5 µl of 10x buffer (100 mM Tris/HCl, 15 mM MgCl2, 500 mM KCl, pH 8·3), 0·5 µl of 25 mM MgCl2, 0·5 µl of 10 mM dNTP mix, 18·25 µl of sterile water, 0·5 µl of each primer (20 µM) and 0·25 µl of Taq polymerase (5 U µl-1) (Roche). The cDNA was amplified with a Perkin-Elmer thermal cycler for 30 cycles. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. The following oligonucleotides were used: hIL-10, 5'-CTGAGAACCAAGACCCAGACATCAAGG-3' and 5'-CAATAAGGTTTCTCAAGGGGCTGGGTC-3'; -actin, 5'-GCTGCGTGTGGCTCCCGAGGAG-3' and 5'-ATCTTCATTGTGCTGGGTGCCAG-3'.
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RESULTS |
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EB1 binds to the hIL-10 promoter in vitro
In order to locate EB1-binding sites, several DNA fragments of the hIL-10 minimal promoter were used in EMSAs together with a purified EB1-His-tagged protein (Fig. 4A). As shown in Fig. 4(B)
, EB1 bound only to DNA fragments III and VI (lanes 8 and 17). On these two fragments more than one retarded complex was visualized, suggesting that these fragments contained more than one EB1-binding site. A 100-fold molar excess of an oligonucleotide harbouring two functional EB1-specific binding sites (ZRE) (Fig. 4B
, lanes 19, 20 and 21), efficiently competed with EB1 binding on probe III and VI (Fig. 4B
, lanes 9 and 18). To further locate the EB1-binding sites on hIL-10 promoter regions III and VI, synthetic oligonucleotides covering these regions were used in an EMSA (Fig. 5A
). As shown in Fig. 5(B)
, EB1 interacted with probe E (lane 1) but very weakly with probe A (lane 2). Accordingly, EB1 interacted with probes B and C (lanes 4 and 6), but weakly with probe D (lane 8), suggesting that at least one EB1-binding site was present in region III. As shown in Fig. 5(C)
, among the oligonucleotides tested encompassing region VI, oligos J, L and G interacted with purified EB1 (lanes 14, 16 and 18 respectively), whereas a very weak binding of EB1 was seen on oligos F and M (lanes 10 and 24 respectively). These results suggested that there are several binding sites for EB1 in the hIL-10 minimal promoter.
EB1 binds to at least five sites in the hIL-10 minimum promoter
Since the purified EB1-His protein bound to oligonucleotides B, C, D, G, J and L in vitro, we characterized precisely the EB1-binding sites in these oligonucleotides by DMS interference (Fig. 6A, B). For oligos B, C and G (Fig. 6A and 6B
respectively), methylation of the guanine residues only partly impaired binding of EB1, indicating that probably more than one EB1-binding site was present on the probes. For oligos J and L (Fig. 6B
), methylation of one or two guanine residues completely impaired binding of EB1, indicating that the residues were important for the in vitro interaction. The results of the DMS interference experiments are summarized in Fig. 6(C)
and the putative EB1-binding sites in the hIL-10 minimum promoter are listed and aligned together with several known functional EB1-binding sites. Methylation interference did not allow to define the EB1DNA contacts on oligos D, F, K and M (data not shown), probably due to very weak binding of EB1 to these DNA probes. Indeed, in oligonucleotides F, M and K, the EB1-binding sites are at the extremity of the double stranded oligonucleotides, and in oligonucleotides F and K, the EB1-binding site is also partially deleted (Fig. 6C
). Taken together these data showed that at least five EB1-binding sites are present in the hIL-10 minimum promoter.
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DISCUSSION |
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Our findings extend the notion that hIL-10 production is activated at different stages of and essential to the EBV infectious cycle. Indeed, hIL-10 is upregulated in latently infected B cells and the viral gene products responsible for this induction are the transmembrane protein LMP-1 (Nakagomi et al., 1994; Vockerodt et al., 2001
) and the EBV-encoded RNAs (EBERs) (Kitagawa et al., 2000
). vIL-10, a viral homologue of hIL-10 encoded by the BCRF1 gene, is also expressed late in the productive cycle (Moore et al., 1990
) and shares a subset of hIL-10 biological activities (Moore et al., 2001
). In addition, we show here that hIL-10 expression was activated by the inducer of the viral productive cycle, EB1, extending considerably previous results demonstrating that hIL-10 production was detected on induction of EBV reactivation (Sairenji et al., 1998
).
On induction of the EBV productive cycle in latently infected B cells, viral gene products are expressed as a result of the transcriptional activity of the EB1 and R proteins. The viral gene EBERs are transcribed during productive infection and the BNLF-1 gene, encoding the LMP-1 protein, is also still expressed (Kieff & Rickinson, 2001). It has been reported that the BNLF-1 gene encodes a truncated form of LMP-1, called lytic LMP-1 (Hudson et al., 1985
), and it has been demonstrated that latent LMP-1 signalling is impaired by lyLMP-1 protein (Erickson & Martin, 2000
). EB1 also interferes with latent LMP-1 signalling since it was recently shown that EB1 completely inhibits the upregulation of MHC class I expression mediated by LMP1 (Keating et al., 2002
). As there is no evidence suggesting that LMP-1 and EBERs are still inducing hIL-10 expression during the productive cycle, one may speculate that EB1-induced hIL-10 expression reflects a selection pressure that imposed the persistence of hIL-10 expression.
We also hypothesize that, at the time of virus reactivation, hIL-10 expression might create a favourable microenvironment for de novo infection of B cells through three different mechanisms: (i) When the productive cycle is initiated, many EBV lytic gene products allow CTL recognition and lysis of the infected cell (Rickinson & Moss, 1997). As described above, EB1 has been associated with a strong decrease of MHC class I molecules at the B cell surface (Keating et al., 2002
). We suggest that EB1-induced hIL-10 confers a broader effect on MHC molecules and thereby on cell types, strongly impairing the cell-mediated immune responses. Indeed, hIL-10 is known to inhibit expression of MHC class II on monocytes (de Waal Malefyt et al., 1991
) and to downregulate MHC class I presentation of antigens (Zeidler et al., 1997
). In this way, hIL-10 suppresses T lymphocyte activities through downregulation of antigen presentation and through direct inhibition of T cell proliferation (de Waal Malefyt et al., 1991
, 1993
). (ii) hIL-10 is also known to downregulate IFN-
production by blood mononuclear cells, by acting on T-helper 1 lymphocytes, monocytes and presumably on dendritic cells in response to stimulation by virus (Moore et al., 2001
; Payvandi et al., 1998
). This biological activity of hIL-10 could be crucial at the time of EBV lytic gene activation of transcription, since it inhibits the host antiviral response and thus protects the infected cell from lysis induced by specific cytotoxic T lymphocytes. (iii) Finally, as hIL-10 is a potent growth factor for B cells (Rousset et al., 1992
), it is reasonable to hypothesize that it might increase the pool of new target cells in vivo through a paracrine pathway.
There is increasing evidence suggesting that EB1 interferes with cellular signalling pathways. Indeed, EB1 is thought to modulate local immune responses as it induces production of the potent immunosuppressive cytokine TGF- (Cayrol & Flemington, 1995
) and inhibits the IFN-
signalling pathway (Morrison et al., 2001
). EB1 also causes dispersal of nuclear PML bodies, which are presumably involved in the regulation of MHC class I antigen presentation (Adamson & Kenney, 2001
). In addition to these previous reports, we have documented a novel function of the viral EB1 protein, induction of hIL-10 gene transcription. Our results, together with those published by others, confirm that IL-10 plays a central role in EBV biology. However, the operation of this role in vivo is not yet clearly understood.
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ACKNOWLEDGEMENTS |
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Received 20 September 2002;
accepted 9 December 2002.