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. 2002 Jul;76(13):6460-72.
doi: 10.1128/jvi.76.13.6460-6472.2002.

Human monocytic cell lines transformed in vitro by Epstein-Barr virus display a type II latency and LMP-1-dependent proliferation

Eric Masy  1 , Eric Adriaenssens, Claire Montpellier, Pascale Crépieux, Alexandra Mougel, Brigitte Quatannens, Gautier Goormachtigh, Nathalie Faumont, Fabienne Meggetto, Claude Auriault, Hervé Groux, Jean Coll
Affiliations

Human monocytic cell lines transformed in vitro by Epstein-Barr virus display a type II latency and LMP-1-dependent proliferation

Eric Masy et al. J Virol. 2002 Jul.

Abstract

Epstein-Barr virus (EBV) classically infects and transforms B lymphocytes in vitro, yielding lymphoblastoid cell lines (LCLs). In contrast to other herpesviruses, EBV is not described as an infectious agent for monocytes. However, recent papers described in vitro infection of monocytes leading to abortive or transient viral expression. In the present study, we report the characterization of E1, a monocytic cell line infected and transformed by EBV. This cell line was derived from an LCL by a drastic electroporation and selection of neomycin-resistant cells, unfavorable to B-cell outgrowth. E1 expressed surface molecules of monocytic lineage (CD14, major histocompatibility complex class II, and CD80) and the c-fms gene, a highly specific marker for the monocytic lineage. This cell line is able to phagocytose and secrete proinflammatory monokines tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8. E1 cells are tumorigenic after injection in nude mice, and a monocytic cell line obtained from one of these tumors (TE1) displayed immunophenotype and functional properties similar to those of E1. We detected the presence of the EBV genome in both cell lines, as well as expression of the EBNA-1 and LMP-1, but not EBNA-2, viral genes, characteristic of a type II latency. LMP-1 influences the phenotype of these monocytic cell lines, as demonstrated by down-regulation of cell proliferation and membrane intercellular adhesion molecule 1 expression due to an LMP-1 antisense strategy. This is the first description of a latently infected human monocytic cell line and the first direct demonstration of an instrumental role for LMP-1 in the proliferation of EBV-transformed cell lines expressing a type II latency.

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Figures

FIG. 1.
FIG. 1.
Membrane markers expressed by E1 and analyzed by flow cytometry. Antibodies used for immunophenotyping are listed in Table 3.
FIG. 2.
FIG. 2.
Detection of c-fms, CSF-1, and c-myc transcripts. Total RNA was isolated from both cells. RNA was reverse transcribed and amplified with pairs of PCR primers specific for the c-fms (A) or CSF-1 (B) gene. Specific products were visualized by ethidium bromide staining on a 1.8% agarose gel. (C) Northern blot experiments performed with total RNA from the cell lines listed and hybridized with c-myc- and GAPDH-specific probes. MW, molecular weight markers.
FIG. 3.
FIG. 3.
E1 and TE1 expressed the EBV genome. (A) Detection of the EBV genome in infected monocytes. The presence of viral DNA in Hirt's extracts was evaluated by PCR amplification of three EBV latent genes: LMP-1, EBNA-1, and EBNA-2. The corresponding PCR products (see primers in Table 2) were visualized by ethidium bromide staining on a 1.8% agarose gel. An LCL was used as a positive control for detection of the viral gene. Lane MW, 100-bp ladder. (B) Detection of viral RNA in E1 and TE1 by RT-PCR and Northern blotting experiments using primers and probes specific for a selected set of viral genes (see primers in Table 2). B95.8 cells were used as a positive control.
FIG. 4.
FIG. 4.
Detection of LMP-1 protein expression in E1 and TE1 cell lines. After fixation and permeabilization, cells were first incubated with anti-LMP-1 antibodies (CS1-4) and then with an anti-mouse Ig labeled with PE. Cells were then analyzed by flow cytometry. Kas B-LCL was used as a positive control. The signal obtained with the isotypic control for E1 is indicated.
FIG. 5.
FIG. 5.
Demonstration of phagocytic properties for EBV-infected monocytes E1 and TE1. E1 and TE1 were incubated with a suspension of E. coli labeled with FITC at 4 (dotted lines) and 37°C (solid lines). After two washes with cold PBS, cells were analyzed on a cytometer at 488 nm. Jurkat cells were used as a negative control, and HL60 cells were used as a positive control. In the center, phagocytosis of E. coli labeled with FITC by E1 cells at 37°C is shown by microscopic fluorescence analysis.
FIG. 6.
FIG. 6.
Kinetics of TNF-α production in culture supernatants of E1 (dark gray bars) and TE1 (light gray bars) after stimulation by PMA (0.1 μg/ml). The TNF-α level was evaluated with an enzyme-linked immunosorbent assay. The U937 cell line (black bars) was used as positive cell control.
FIG. 7.
FIG. 7.
Inhibition of proliferation of EBV-positive cells by an LMP-1 antisense oligonucleotide. Proliferative responses were evaluated by measuring [3H]thymidine uptake after 48 h of cell culture. Cell lines TE1, Rafa B-LCL, and DG 75 were cultured alone or with oligonucleotide AS2 (▪) or SC2 (□) at two concentrations (5 and 10 μM). Results were expressed as percentages compared with that for the untreated control. The proliferative rate of untreated cells was arbitrarily defined as 100%. The values represent the means of triplicate assays ± standard deviations.
FIG. 8.
FIG. 8.
Effect of LMP-1 inhibition on expression of different markers in E1 or TE1 cells. (A) Effect on ICAM-1/CD54 expression on E1 cells analyzed by flow cytometry. (Top) The LMP-1 antisense oligonucleotide (AS2) down-regulated membrane expression of LMP-1 molecular target ICAM-1 on E1 cells. Scramble oligomers were used as the control (SC2). (Bottom) The same results were obtained with cells cotransfected with pEGFP-C1 and pSV LMP-1-AS or with pcDNA3 containing the coding sequence for mutated IκBα32/36A. The percentage of ICAM-1-expressing cells and the density of expression (MnI X) in these conditions are indicated. (B) Western blot analysis of several markers after LMP-1 inhibition experiments with TE1 cells. Immunoblots were probed with the S12 monoclonal antibody (LMP-1) or antibodies specific for TRAF-2, BCL-2, and actin proteins. Detection was performed after antisense oligonucleotide (AS2) or scramble (SC2) treatment, and results were compared to those for untreated cells (U). (C) Western blot and cytometry analyses of monocytic markers. (Left) Detection of CSF1-R after AS2, SC2, or PMA treatment in TE1 cells. U937 cells untreated or stimulated by PMA were used as the control for CSF1-R induction. (Right) Flow cytometry profile for CD14 labeling after treatment of TE1 cells. Rafa B-LCL is shown as a negative control.
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