Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers - PubMed (original) (raw)

Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers

Dirk M Pegtel et al. J Virol. 2004 Nov.

Abstract

Epstein-Barr virus (EBV) is found frequently in certain epithelial pathologies, such as nasopharyngeal carcinoma and oral hairy leukoplakia, indicating that the virus can infect epithelial cells in vivo. Recent studies of cell lines imply that epithelial cells may also play a role in persistent EBV infection in vivo. In this report, we show the establishment and characterization of an ex vivo culture model of tonsil epithelial cells, a likely site for EBV infection in vivo. Primary epithelial-cell cultures, generated from tonsil explants, contained a heterogeneous mixture of cells with an ongoing process of differentiation. Keratin expression profiles were consistent with the presence of cells from both surface and crypt epithelia. A small subset of cells could be latently infected by coculture with EBV-releasing cell lines, but not with cell-free virus. We also detected viral-DNA, -mRNA, and -protein expression in cultures from EBV-positive tonsil donors prior to in vitro infection. We conclude that these cells were either already infected at the time of explantation or soon after through cell-to-cell contact with B cells replicating EBV in the explant. Taken together, these findings suggest that the tonsil epithelium of asymptomatic virus carriers is able to sustain EBV infection in vivo. This provides an explanation for the presence of EBV in naso- and oropharyngeal pathologies and is consistent with epithelial cells playing a role in the egress of EBV during persistent infection.

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Figures

FIG. 1.

Tonsillar explant culture scheme. Cultures were grown as described in Materials and Methods. The procedure involves three separate steps. In step 1, tonsillar explants are placed beneath tissue culture transwell inserts. The membrane is porous, and a weight is placed on top of the insert membrane. DMEM-F12 containing 10% FBS and 1 mM Ca2+ was used during the first 4 days of culture. In step 2, the inserts are removed and the culture medium is changed to a low-calcium defined serum-free medium with keratinocyte growth factor (Gibco). This ensures the outgrowth of epithelial cells, abolishes fibroblast growth, and kills lymphocytes. In step 3, the explants are removed and epithelial cells are transferred to new dishes and culture slides. Photographs of the cultures at different stages are shown. Epithelial cells begin to migrate out of the tissue explants (T) on day 4 (d4) (black arrow). On day 7, large colonies surround the tissue explants with no visible indication of surviving lymphocytes. On day 7, large colonies (>1 cm in diameter) of cells with typical epithelial morphology surround the explants. By day 10, adherent cells have different morphologies, some cells having acquired a very large cytoplasm-to-nucleus ratio (white arrow), often seen in differentiated squamous epithelial cells. Thus, it appears that an active epithelial differentiation process is ongoing in the cultures.

FIG. 2.

HC and IF analyses of primary tonsil epithelial cells indicate cellular heterogeneity. Primary tonsillar epithelial cells (days 7 to 10) were grown to confluence on chambered culture slides. Immunostaining was performed with a panel of monoclonal antibodies against epithelial (keratin, involucrin, and ESA) markers and a B-cell marker, CD21. Positive staining is red. For HC, counterstaining was performed with hematoxylin and eosin (blue.) For IF, nuclei are stained with DAPI (4′,6′-diamidino-2-phenylindole) (blue). Larger cells (arrows) are negative for ESA (Ber-ep4 clone) and positive for involucrin. This confirms that the phenotypic differences are linked to the differentiation stage.

FIG. 3.

Primary tonsillar epithelial cells are susceptible to in vitro infection with EBfaV-GFP by cell-cell contact but not with cell-free virus. (A) GFP expression could be detected in epithelial cells as early as 24 h postinfection (white arrows). Occasional adherent producer B cells are indicated with white arrowheads. Left panels, bright field; right panels, fluorescence for the same field of cells. (B) GFP-expressing cells are cytokeratin positive (indicated by white arrows). Left panels, red fluorescence for cytokeratins; right panels, green fluorescence for GFP expression on the same field of cells. (C) Infection by cell-free virus was never observed with epithelial cells, but Daudi cells could be efficiently infected (36 h postinfection) with EBfaV-GFP viral supernatant; the infection efficiency was dose dependent.

FIG. 4.

In vitro infection of primary epithelial cells. Seven- to 10-day epithelial cells were incubated in a 1:1 ratio for 1 to 3 days with viral producer B cells stimulated to produce virus. (A and B) Merged immunofluorescence images of EBfaV-GFP-infected epithelial cells are cytokeratin positive (red) and express GFP (green), which appears as yellow; nuclei are stained with DAPI (4′,6′-diamidino-2-phenylindole) (blue). (C) Involucrin-positive cells are infected with EBfaV-GFP. (D to I) Primary tonsil epithelial cells infected in vitro with B95.8 EBV are latently infected. Isolated cells with epithelial morphology coexpress LMP1 (red) (D and G) and EBNA1 (green) (E and H), indicating a latent stage of infection in these cells. Nuclei are stained with DAPI (blue). Merged images (F and I) show the nuclear location of EBNA1 (light blue) and the cytoplasmic location of LMP1.

FIG. 5.

RT-PCR for LMP1 expression in primary ex vivo epithelial cells from the palatine tonsil. RT-PCR was performed on RNA from 2 × 105 to 1 × 106 day 10 to 14 epithelial cells. The PCR products were fractionated on agarose gels, Southern blotted, and hybridized with an LMP1-specific probe. The size of the PCR product (150 bp) is indicated. Limiting dilution was performed with serially diluted EBV-positive IB4 cells as a positive control (+). EBV-negative BJAB cells and H2O were negative controls (−). Epithelial cultures of 15 individual tonsils are shown; 12 were from EBV-positive donors (T), 9 of which were positive for LMP1, and 3 were from EBV-negative donors (T*), none of which were positive for LMP1.

FIG. 6.

LMP1 protein expression in ex vivo tonsillar epithelial cells. Epithelial cells were cultured as described in Materials and Methods and stained between days 7 and 10 with the anti-LMP1 monoclonal (S12) primary antibody for HC or IF. (A) LMP1 expression was assessed in the EBV-positive control B-cell line IB4. (B) Epithelial-cell culture from an EBV-negative tonsil. (C and D) Epithelial-cell cultures from EBV-positive tonsils. Left panel, low power; right panel, high power. (E and F) Epithelial-cell cultures from EBV-positive tonsils analyzed by HC (E) or IF (F). (G and H) Epithelial-cell cultures from EBV-negative tonsils infected in vitro and analyzed by HC (G) or IF (H). Note the similarity in staining observed when LMP1-expressing epithelial cells from an EBV-positive tonsil (ex vivo [E and F]) are compared with epithelial cells from an EBV-negative donor infected in vitro (in vitro infection [G and H]). Panel F shows a typical epithelial perinuclear distribution of LMP1 in an ex vivo-infected cell.

FIG. 7.

(A to I) Immunofluorescence images of lytic gene expression in primary cultures from two tonsils. Cells were cultured in the presence of acyclovir until day 4. On day 5, SFM plus KGF was added, and the cells were transferred on days 7 and 8 to culture slides. Cytospins of 8- to 16-h IgG-treated Akata cells were used as positive controls (J to M). VCA staining is cytoplasmic (J), whereas Zebra (Z) and early antigen (Ead) staining is nuclear (K and L). Epithelial cells were fixed and stained on day 10 with the pooled monoclonal antibodies against Ead, Z, and VCA. The antibodies were pooled to increase sensitivity (M). The white arrows in panels B and C point to cytoplasmic VCA staining. Merged images are presented to emphasize the predominantly nuclear nature of the positive staining in both cultures derived from Z and Ead (D to L).

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Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers - PubMed (original) (raw)