Epstein-Barr Virus Infection in Ex Vivo Tonsil Epithelial Cell Cultures of Asymptomatic Carriers Dirk M

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Epstein-Barr Virus Infection in Ex Vivo Tonsil Epithelial Cell Cultures of Asymptomatic Carriers Dirk M. Pegtel,1 Jaap Middeldorp,2 and David A. Thorley-Lawson1* Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts,1 and Department of Pathology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands2

Received 18 April 2004/Accepted 17 May 2004

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.

Epstein-Barr virus (EBV) is a ubiquitous, persistent human tion of oropharyngeal epithelial cells was provided when ␤1 herpesvirus whose tropism for B lymphocytes is well estab- integrin was identified as a crucial component of the receptor lished. However, some reports have also suggested an epithe- that mediates EBV infection in subsets of oral epithelial cell lial-cell tropism for the virus based on claims of having iden- lines (54). Other in vitro findings provided a mechanistic ex- tified EBV-infected pharyngeal epithelial cells in vivo (46). planation for this proposed dual tissue tropism (8, 10). Virions This led many investigators to propose that the mucosal epi- produced by B cells in vitro have glycoprotein expression pat- thelium of the nasopharyngeal region was the site of EBV terns that favor infection of epithelial cells and vice versa. This persistence and that the infection of B cells was not critical for could result in a preferential exchange of EBV between the viral persistence (1, 41, 42, 61). Subsequently, however, these tissues. If EBV in vivo is transmitted back and forth between findings were challenged based on the failure of other investi- epithelial cells and B cells, the tissues must be in close prox- gators to reproduce them (25, 35) and the mounting body of imity to one another. Tonsil reticulated crypt epithelium (37, evidence implicating memory B cells as the site of EBV per- 38) is one such site where EBV-infected B cells are in intimate sistence (4, 13, 17, 51, 52). Specifically, it has not been possible contact with neighboring epithelium (2, 34). Great improve- to reproducibly detect infected epithelial cells in cross sections ments have been made in understanding the role of B cells in of tonsils or exfoliated epithelial cells in the saliva of acutely EBV persistence in vivo due to the accessibility of infected infected patients (25, 35). Even though these studies did not primary B lymphocytes in the blood and lymph nodes (51, 52) rule out low levels of infected cells, they encouraged the belief and the availability of an in vitro infection system (26). This that EBV does not normally infect healthy epithelial cells in work has established the importance of B cells in the tonsil vivo. Nevertheless, EBV is 100% associated with naso- and lymphoepithelium as a critical site for the establishment of oropharyngeal epithelial pathologies, such as nasopharyngeal latent persistent infection and for replication of the virus (51). carcinoma (NPC) (3) and oral hairy leukoplakia (OHL) (18). In contrast, evidence that nasopharyngeal epithelial cells play In NPC, the epithelial cells are predominantly latently infected, whereas in OHL, an AIDS-associated lesion of the oral a role in EBV persistence and/or replication in healthy carriers epithelium, infectious virus is produced by epithelial cells in is still lacking. large amounts (58). From these findings, it is clear that EBV One possible role for the epithelium in the nasopharynx is as can latently infect and replicate in oral- and nasopharyngeal the first tissue that becomes infected after exposure to the epithelia in vivo. This raises the possibility that these lesions virus, because EBV is transmitted via saliva (19). Lytic repli- may be amplified versions of infected healthy epithelium. cation in epithelial cells could then act as a transit route or an A recent in vitro breakthrough in understanding the infec- amplification step prior to infection of the main target, B cells residing in the underlying lymphoepithelium. Alternatively, epithelial cells could be used to transit and release virus, pro- * Corresponding author. Mailing address: Department of Pathology, duced from infected B cells in the lymphoepithelium, back into Jaharis Bldg., Tufts University School of Medicine, 150 Harrison Ave., Boston, MA 02111. Phone: (617) 636-2726. Fax: (617) 636-2990. E- saliva. This kind of dual tissue tropism strategy is consistent mail: [email protected]. with the behavior of other ubiquitous herpesviruses (43), and

12613 12614 PEGTEL ET AL. J. VIROL. in vivo evidence for such an infection route has recently pipette. The serum-free culture medium was then changed every 2 days. Culture been provided for simian immunodeficiency virus in mon- dishes with contaminating spindle-like fibroblasts were discarded. keys (50). All cell lines were maintained under standard conditions with 5% CO2 at 37°C in a humidified atmosphere. IB4, B95-8 (a gift of E. Kieff), BJAB, Akata, and The failure to detect infected epithelial cells in vivo and the Daudi (all American Type Culture Collection) cell lines were maintained in lack of an accurate primary in vitro nasopharyngeal epithelial complete RPMI 1640 medium supplemented with 10% fetal bovine serum, culture model has greatly hampered research in understanding penicillin plus streptomycin, and L-glutamine. EBfaV-GFP (green fluorescent the role these cells might play in persistence. For these rea- protein) cells were maintained in complete RPMI with 100 ␮g of G418 (Gibco)/ sons, we set out to establish and characterize a primary tonsil ml. EBfaV-GFP (a gift of Richard Longnecker) is a derivative of the B95-8 virus-producing cell line in which the viral LMP2 gene is replaced by a gene epithelial culture system that retains the heterogeneity of the cassette consisting of the enhanced-GFP gene driven by the cytomegalovirus tissue found in vivo. We have developed early ex vivo tonsil promoter and the neomycin resistance gene driven by the simian virus 40 pro- epithelial-cell cultures that express keratin markers indicative moter (48). of crypt and surface tonsil epithelia. A subset of these cells is PCR. Reverse transcription (RT)-PCR for latent genes was performed as ϫ 5 reproducibly infectible in vitro by coculture with EBV producer previously described (5, 20). Briefly, RNA was purified from 2 10 epithelial cells and 5 ϫ 106 lymphocytes using Trizol reagent (Invitrogen) as described by cells. Surprisingly, evidence of EBV infection was detected prior the manufacturer. Lymphocytes from EBV-negative tonsils, the EBV-negative to the addition of exogenous virus in cultures from EBV-positive BJAB B-cell line, and the EBV-positive IB4 B-cell line were used as controls. tonsils even when maintained in the presence of acyclovir. These cDNA was prepared as described previously (5, 20), except that the 20-␮l cDNA observations suggest that the epithelial cells were infected ei- mixture was not ethanol precipitated but brought up to 100 ␮l with high-perfor- ther in vivo or rapidly after explantation by virus already mance liquid chromatography H2O and used directly. PCR was performed on the synthesized cDNA for EBNA1U-K, EBNA1Q-K (EBNA1 from the Qp present in the tissue explants. To our knowledge, these findings promoter), EBNA2, LMP1 (latent form), and LMP2a. The reaction was carried are the first to describe EBV infection of healthy tonsil epi- out in a final volume of 50 ␮l in a mixture consisting of 50 mM KCl, 20 mM Tris thelial cells derived from asymptomatic virus carriers. Our data (pH 8.4), 2.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphates, and 20 pM support the model of dual epithelial-lymphoid tropism for the (each) amplimers (final concentrations). The exception was LMP1, for which 3.0 virus in vivo and the possibility that healthy tonsil epithelium in mM MgCl2 was used. The amplimers and PCR strategies are described in detail elsewhere (5, 20). The PCR products were visualized by Southern blotting as vivo may play a role in transmission of the virus as part of the described previously (33). Probes for blotting were made from PCR products viral life cycle and suggest that EBV can play an initiating role derived from the IB4 cells. in associated epithelial lesions, such as NPC and OHL. Coculture and in vitro infection. EBfaV-GFP cells and B95-8 cells were treated with 20 ng of tetradecanoyl phorbol acetate (Sigma)/ml and 3 mM butyric acid (Sigma) for 1 to 4 days, while Akata cells were cross-linked with 100 ␮gof MATERIALS AND METHODS Fab immunoglobulin G (IgG) (Jackson Laboratories)/ml overnight to induce Primary cultures and cell lines. Tonsillectomies were performed at Massa- virus release. Cell suspensions were centrifuged (5 min at 3,000 ϫ g), and the chusetts General Hospital on 6- to 14-year-old patients because of obstructed- supernatants were filtered through a 0.45-␮m-pore-size filter. One milliliter of ϫ breathing disorders. Tonsils were kept on ice in PBSAPSF (1 phosphate- supernatant (cell-free virus) was added to 2 ϫ 105 epithelial cells in six-well buffered saline [PBS]–0.5% bovine serum albumin–PennStrepFungizone; Gibco) plates in 3 ml of medium. Daudi cells were incubated for1hinundiluted and and were processed between 2 and 6 h after surgery. The epithelial culture 10-fold-diluted supernatants, washed, and placed in culture. For infection method we applied is a modified version of the method for culturing epidermal through cell-to-cell culture, producer cells were stimulated overnight, washed, keratinocytes developed by Lindberg and Rheinwald, Rheinwald and Green, and and resuspended in SFM. Virus-releasing cells were added to six-well plates and Schon and Rheinwald (30, 40, 44). Epithelial sections of the tonsil exterior were incubated with 1 ϫ 105 to 2 ϫ 105 day 7 to 10 subconfluent layers of primary removed with surgical tools. Connective tissue sections of the interior were epithelial cells at a 1:1 or 2:1 ratio. Floating cells were removed at various time separated from soft tissue (lymphocytes) by scraping the blunt side of a razor points (days 1 to 3) postinfection by washes, and epithelial cells were transferred blade over the tissue sections. The tissue sections were washed in cold PBSAPSF to culture slides 4 days postinfection and prepared for staining. at least 10 times, minced with razorblades, and extensively washed in cold Staining. Cells were cultured as described and replated at various time points PBSAPSF to remove nonepithelial stroma (mostly lymphocytes) and debris. The (typically days 7 to 10) in two- and eight-well chambered culture slides (Costar) lymphocytes were isolated from the supernatant by Ficoll-Hypaque (Amersham in SFM. Cytospins of IB4 (an EBV-positive cell line) and BJAB (an EBV- Pharmacia) centrifugation, washed two times with 1ϫ PBS–0.5% bovine serum negative cell line) cells were used as controls for viral-protein detection and albumin, and resuspended at 2 ϫ 107/ml. Small explants of 1 to 2 mm3 were B-cell (CD21) and epithelial marker (Ber-ep4) expression. Cytospins and culture resuspended in 10% fetal bovine serum (FBS)–PennStrepFungizone–Dulbecco’s slides were treated the same way in all staining procedures. For immunohisto- modified Eagle’s medium (DMEM)-F12–1 mM calcium (Gibco) (high-calcium chemistry (HC), cells were fixed and permeabilized for 10 to 15 min with 100% serum-containing medium), washed twice, and placed in a 100-mm-diameter dish chilled acetone. For immunofluorescence (IF), cells were fixed and permeabil- and incubated overnight at 37°C in an incubator (day zero). This dramatically reduced the occurrence of fungal and/or bacterial contamination. For ex vivo ized for 30 min at room temperature in 4% paraformaldehyde, followed by 10 infection experiments, 0.1 mM acyclovir (UDL Labs, Rockford, Ill.) was added min with 0.1% Triton X-100 in phosphate-buffered saline (PBS) at 4°C. The to the medium. The next day, tissue sections were washed in medium again to slides were subsequently blocked with appropriate serum for1hatroom tem- remove the remaining lymphocytes, and transwell devices (24-mm diameter; perature prior to indirect staining. For indirect IF staining of viral proteins, the Costar) were placed on top of ϳ30 to 40 explants. An inert sterile stirring magnet cells were incubated with primary monoclonal antibodies: LMP1-S12 (dilution, was placed in the transwell for two reasons: to keep the explants attached to the 1:5,000), EBNA1, viral capsid antigen (VCA), and EAD (dilution, 1:1,000) (gifts bottom of the plate and to stimulate epithelial-cell migration out of the explants. of J. Middeldorp) and BZLF1 (dilution, 1:10) (Dako) at room temperature for Serum-containing medium was added in and surrounding the transwells. Three 30 min and then washed three times in PBS. Secondary goat anti-mouse Alexa transwells were placed in one 100-mm-diameter culture dish. The medium was 488 and Alexa 594 antibodies (dilution 1:5,000) (Molecular Probes, Breda, The replaced on day 2. On days 3 and 4, depending on individual culture growth, the Netherlands) were incubated for1hinthedark. The slides were washed with transwells were gently removed and discarded. The medium was replaced by PBS (three times) and examined with a fluorescence microscope (Nikon E400). defined keratinocyte serum-free medium (SFM) plus keratinoctyte growth factor For HC and IF of cellular proteins, primary antibodies to keratin 8 (K8), K18, (KGF) (Invitrogen) containing 0.1 mM Ca. Most tissue explants remained con- K8/K18, K10, K19, K13, involucrin, fillagrin, CD21, and AE1/AE3 (pankeratins) nected to the bottom of the plate. Fresh medium was added each day after gentle (Neomarkers, Fremont, Calif.) were typically diluted 1:200 and incubated for 45 washing. Typically, around days 7 to 10 in culture, colonies 1 cm in diameter min at room temperature. For HC, the slides were developed with an LSAB surround the tissue explants. If the explants were removed when the colonies detection kit (Dako) used according to the manufacturer’s instructions, and the were less than ϳ0.2 cm in diameter, further colony growth was markedly cur- slides were visualized using a light microscope (Nikon TE200). For IF, the slides tailed. Tissue explants were gently removed by suction with a vacuum Pasteur were developed as described above. VOL. 78, 2004 EBV-INFECTED EPITHELIAL CELLS IN TONSILS 12615

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 Ca2ϩ 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 (Ͼ1 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.

RESULTS can be distinguished based upon expression of differentiation markers and morphology (30, 32). A subset of morphological Culture and characterization of primary tonsil epithelial large epithelial cells appeared in the cultures over time (Fig. 1, cells. The culture process was performed in three phases. The day 7 versus day 10). These cells were epithelial surface antigen first culture phase, DMEM-F12 plus 10% FBS with a high (1 (ESA) negative (early differentiation marker [6]), involucrin mM) calcium concentration, is designed to stimulate the mi- positive (late differentiation marker [56]), and keratin 13 neg- gration and adherence of epithelial cells out of the small tonsil ative (early differentiation marker) and had a large cytoplasm/ tissues to the culture dish. The second phase, SFM with low nucleus ratio (Fig. 2E, I, and J). This expression profile is con- (0.1 mM) calcium plus defined KGF, ensures the elimination sistent with differentiated squamous epithelial cells (32, 56), of lymphocytes and fibroblasts through cell death but promotes epithelial-cell viability and growth. In this phase, keratinocyte suggesting that a process of terminal differentiation was ongo- growth and migration are still supported by the existing tissue ing in the cultures. Occasionally epithelial cells tended to explants (40). In the third phase (SFM plus KGF), the explants round up spontaneously and detach from the dish, a common are gently removed and the epithelial cells are replated to behavior of terminally differentiating primary epithelial cells in another dish at ϳ60% confluency. Cells with clear epithelial vitro (9). Positive staining of these cells for involucrin con- morphology started to migrate on days 3 to 5 in culture, and on firmed that they were differentiated (data not shown). days 5 to 8, colonies of various sizes surrounded most individ- Most epithelial tissues are complex, and individual keratin ual tissue explants. The details are described in Materials and protein expression can be used to define the origin of epithelial Methods. Figure 1 shows a schematic and representative pho- cells (30). Tonsil epithelium is comprised of stratified surface togaphs of the three culture phases. In general, the primary and reticulated crypt epithelia (11, 38). In situ staining of tonsil epithelial cells remained viable for up to 4 weeks in culture. epithelium suggests that expression of the simple epithelial To characterize the purity, heterogeneity, and differentiation keratins K18 and K8 is limited to the crypt epithelial cells (11). stage of the cultured ex vivo tonsil epithelial cells, we per- We detected variable K8 and K18 expression early in our ex formed HC and IF staining for epithelial and lymphoid mark- vivo tonsil cultures, suggesting that a subpopulation of epithe- ers. Day 7 to 8 cultures were isolated and plated at ϳ80% lial cells in our cultures were of crypt epithelial lineage (Fig. confluency on culture slides and used for staining the next day. 2D and H). In addition, we detected variable K19 expression, One hundred percent of the cells stained positive for AE1/AE3 another potential tonsil crypt lineage marker (11). The variable (pankeratinocyte marker) but were completely negative for expression of K19 in the cultures was particularly apparent by CD21 (pan-B-cell marker) (Fig. 2A, B, and C), indicating that immunofluorescence staining compared with pancytokeratin the cultures were of ϳ100% epithelial lineage. Similar results expression (Fig. 2A, B, F, and G) (11, 32). In summary, our were obtained when the cells were analyzed by flow cytometry. culture method produces heterogeneous squamous epithelial The absence of keratin 10 and fillagrin expression as early as cultures in which differentiation appears to be ongoing and day 7 and later (not shown) indicated that the cells had not keratin expression patterns are consistent with those found in undergone growth arrest and were of the nonkeratinizing squa- tonsil epithelial cells in situ (11, 32). These results are summa- mous epithelial lineage (32). Individual epithelial cell types rized in Table 1. 21 ETLE AL. ET PEGTEL 12616

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 (4Ј,6Ј-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. .V J. IROL . VOL. 78, 2004 EBV-INFECTED EPITHELIAL CELLS IN TONSILS 12617

TABLE 1. Summary of phenotypic characterization of primary In vitro infection. If tonsil epithelium is important in EBV epithelial cultures from the palatine tonsil pathogenesis, the cells should be infectible with EBV. Cocul- Range of ture with EBfaV-GFP-expressing EBV-producing cells (49), Marker Cells expression levelsa but not incubation with infectious viral supernatant, resulted in Keratins infection of all (10 of 10) independent epithelial cultures tested Pankeratins ϩ/ϩϩ All (Fig. 3). This is consistent with earlier findings that cell-free Keratin 19 ϩ/ϩϩ Subset virus is usually incapable of or inefficient at infecting most Keratin 8/18 ϩ Subset ϩ epithelial cells in vitro (16, 23, 45). The infection frequency Keratin 13 Subset ϳ Keratin 10 Ϫ None with EBfaV-GFP was estimated to be 0.01 to 0.5% of the Keratin 8 ϩ Subset total cell population by counting GFP-expressing cells with Keratin 18 ϩ Subset epithelial morphology on day 3 postinfection. To test the possibility that the titer of cell-free virus was too low, we at- Other ϩ tempted to infect Daudi cells with the cell-free EBfaV-GFP ESA Majority ϳ Involucrin ϩϩ Subset virus. We observed infection levels of up to 70% in Daudi Fillagrin Ϫ None cells with the same cell-free virus supernatant that was unsuc- CD21 Ϫ None cessful at infecting primary epithelial cells (Fig. 3C). This in- a ϩ, clearly positive staining; ϩϩ, strong positive staining; Ϫ, no staining. dicates that susceptibility to infection, rather than viral titers, is Variable expression of specific keratins in subsets of cells indicates a heteroge- the limiting factor. neous population of epithelial cells derived from tonsil crypt and surface epithe- To prove that the infected cells were of epithelial origin, lium. they were stained for cytokeratin expression (Fig. 3B and Fig. 4A and B). True infected epithelial cells (Fig. 3A and B) could be distinguished from occasional adherent EBV producer lym-

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. 12618 PEGTEL ET AL. J. VIROL.

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 (4Ј,6Ј-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. phoid cells (Fig. 3A and B) based on their extremely bright signal was derived from infected cells or from virions, virus GFP levels, morphology, and lack of keratin expression. Dual fragments, or cell debris from infected cells released by the imaging for cytokeratins and involucrin suggests that both un- tonsil explants. To distinguish between these possibilities, we differentiated and large differentiated epithelial cells could be performed RT-PCR analysis for a range of viral genes, includ- infected (Fig. 4A to C). ing EBNA1U-K, EBNA1Q-K, EBNA2, LMP1, and LMP2 We next tested whether latent viral proteins are expressed genes. To our surprise, RNA for LMP1 and only LMP1 was and if susceptibility to infection is perhaps limited to the arti- robustly and routinely detected in the cultures from 21 of 30 ficial EBfaV-GFP virus strain, which lacks LMP2 (49). We EBV-positive tonsils but in 0 out of 15 cultures from EBV- cocultured the epithelial cells with phorbol myristate acetate- negative tonsils. Limiting dilutions of Southern-blotted LMP1 butyric acid-stimulated B-95.8 marmoset and IgG-stimulated RT-PCR products (Fig. 5) illustrate that transcript levels in Akata (not shown) cell lines and performed IF staining for 2 ϫ 105 to 1 ϫ 106 epithelial cells were equivalent to 10 to 100 cytokeratins, EBNA1, EBNA2, LMP1, and LMP2. Both virus control EBV-infected IB4 B cells. By comparison, signals for strains were able to infect epithelial cells, as judged by the the other genes were detected in only one or two cultures at or detection of EBNA1 and LMP1 protein expression within the close to the detection limit, casting doubt on the significance of same cells (Fig. 4D to I). EBNA2 and LMP2 expression was these findings. LMP1 is expressed both in latently infected cells not detected. These results indicate that latent genes are ex- and in cells replicating the virus. However, the replicative form pressed in the EBV-infected primary tonsil epithelial cells and is truncated and lacks exon 1. The PCR primers used were that infection is not strain specific. designed to bridge exons 1 and 2 and therefore detect only the Ex vivo infection. During the course of characterization and latent form of LMP1 (53). The reproducible detection of the infection of the primary epithelial cells, we observed that EBV latent form of LMP1 mRNA demonstrated that the primary DNA was routinely detectable in the cultures prior to infection cultures contained latently infected cells. (data not shown). Since we employed highly sensitive PCR to It is unlikely that the LMP1 transcript signals we detected detect the viral DNA, we could not distinguish whether the were from viable infected B cells because the cultures were VOL. 78, 2004 EBV-INFECTED EPITHELIAL CELLS IN TONSILS 12619

washed frequently with medium to remove nonadherent cells and the epithelial cells were split twice before analysis by gently washing them with trypsin prior to transfer to 100-mm-diameter dishes. By that time, no B cells could be detected in the cultures by histochemistry or flow cytometry. Moreover, the tissue culture conditions were highly selective against lymphocytes and other nonepithelial cells. Control experiments with primary B cells or EBV-transformed lymphoblastoid cell lines showed that all the B cells were dead after 2 to 3 days of culture in the selective medium (data not shown). This raised the possibility that there were latently infected epithelial cells in the cultures. To identify which type of cell was infected, we FIG. 5. RT-PCR for LMP1 expression in primary ex vivo epithelial performed immunochemistry and fluorescence staining to de- cells from the palatine tonsil. RT-PCR was performed on RNA from tect LMP1 protein expression (Fig. 6). We chose LMP1 be- 5 6 2 ϫ 10 to 1 ϫ 10 day 10 to 14 epithelial cells. The PCR products were cause the gene was the only one that we found to be repro- fractionated on agarose gels, Southern blotted, and hybridized with an LMP1-specific probe. The size of the PCR product (150 bp) is indi- ducibly expressed at the transcript level in the cultures. In cated. Limiting dilution was performed with serially diluted EBV- control experiments, we were able to reliably detect a single positive IB4 cells as a positive control (ϩ). EBV-negative BJAB cells EBV-positive LMP1-expressing cell in a background of 105 Ϫ and H2O were negative controls ( ). Epithelial cultures of 15 individ- EBV-negative cells, indicating the sensitivity of this staining ual tonsils are shown; 12 were from EBV-positive donors (T), 9 of (data not shown). In double-blind studies, we detected LMP1 which were positive for LMP1, and 3 were from EBV-negative donors (T*), none of which were positive for LMP1. protein expression in single cells with clear epithelial-cell morphology from 5 of 19 independent 7- to 10-day-old primary epithelial cultures. Subsequent analysis revealed that all five

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. 12620 PEGTEL ET AL. J. VIROL. also expressed LMP1 RNA, based on RT-PCR for latent DISCUSSION LMP1 transcripts, and were derived from EBV-positive tonsils, based on DNA PCR (Fig. 6C to H), whereas 10 LMP1-nega- The results presented in this paper relate to three important tive cultures were derived from EBV-negative tonsils (Fig. 6B). areas of EBV interaction with epithelial cells. These are the Thus, LMP1-expressing epithelial cells were found in cultures establishment of a representative primary culture system for a from 5 of 9 EBV-positive tonsils and 0 of 10 EBV-negative hypothesized target of infection in vivo (tonsil epithelium), the demonstration that a small subset of such cells is infectible in tonsils. The level of infection based on LMP1 protein staining vitro by exogenous virus via cell-cell contact, and the observa- was ϳ5to10per105 epithelial cells (0.005 to 0.01%), quite tion that, without adding any exogenous virus, EBV-infected similar to the levels of infection reported for the B-cell com- epithelial cells are already present in cultures from EBV-pos- partment (27). LMP1 (latent) transcripts were detected in all itive individuals. the cultures where LMP1 protein was found. We were unable Tonsil epithelium is a heterogeneous lymphoid organ con- to find evidence of EBNA1, EBNA2, or LMP2 expression in taining two different types of actively differentiating epithelia, any epithelial cells. The absence of EBNA1 may imply that the the lining stratified squamous epithelium and the reticulated infected cells were not proliferating (12), and this would be crypt, or lymphoepithelium. The tonsil crypts represent a spe- consistent with the observation that the LMP1-positive cells cialized compartment, important in the immunological func- were always found alone, never in clusters. However, as with all tions of the tonsil because of the immediate proximity of the histochemical and immunofluorescence studies, it is impossible epithelial and lymphoid tissues (38). Human immunodefi- to resolve whether the failure to find a protein represents ciency virus has been reported to specifically target this region, absence of the protein or whether the levels of expression were possibly because of the close interaction of human immuno- too low to be detected. deficiency virus targets (15). Keratin expression patterns con- One possible explanation for the presence of these infected firmed that our procedure generated heterogeneous epithelial- epithelial cells comes from the well-known phenomenon that B cell cultures. K8, K18, and K19 expression in subsets of cells cells latently infected with EBV will replicate the virus when suggests that they were derived from the reticular crypt epi- placed in culture. It was conceivable, therefore, that the epithelium, and the appearance of large differentiated cells was thelial cells became infected through cell-to-cell contact with consistent with an ongoing active process of differentiation in such cells. This interpretation is unlikely, because the explant the cultures (11, 32). It is possible that the patterns of keratin cultures were continuously maintained in the presence of a expression we observed were induced in vitro. However, pri- high concentration of acyclovir (0.1 mM) in the media. Acy- mary oral epithelial cells possess intrinsically determined pro- clovir is a common antiviral agent (29) that effectively blocks grams for keratin expression, which are believed to be unaf- viral replication with a 50% effective dose of 0.3 ␮M, based on fected by short-term in vitro culture (30). Interestingly, early blocking viral replication in a lymphoblastoid cell line in vitro. removal of the tissue explants abolished the growth and via- We conclude, therefore, that there are bona fide LMP1-posi- bility of the remaining adherent epithelial cells. It is conceiv- tive, latently infected epithelial cells in the cultures. able that culturing epithelial cells from tissue explants provides Viral replication is a frequently reported consequence of a desirable site-specific mesenchymal influence in initial stages EBV infection of epithelial cells in culture which might be of culture. Mesenchymal cells are absent from cultures in differentiation dependent (10, 22, 23, 28, 47, 54). Since our which epithelial cells are isolated from enzymatically digested cultures were routinely grown in the presence of acyclovir, it tissues. This culture procedure often requires an artificial would not be possible to detect the presence of viral replica- feeder layer, which might benefit the growth of particular sub- tion. Therefore, we derived epithelial cell cultures in which populations of epithelial cells (24, 31). Our culture dynamics support the notion that primary epithelial cells are sensitive to acyclovir was removed after day 4. The cells were further their milieu (i.e., stroma), which is likely responsible for the incubated for 4 to 6 days in the absence of acyclovir in selective contextual specificity of various epithelia (37, 40). media. The epithelial cells were then stained with a cocktail We consistently detected EBV DNA, RNA, and proteins in of antibodies recognizing immediate-early (BZLF1), early the cultures. The question arises as to the origin of these (EAD), and late (VCA) viral lytic proteins. Lytic antigen ex- infected cells. The obvious explanation is that we were detect- pression was detected in two of two cultures from EBV-posi- ing contaminating infected B lymphocytes. There are several tive tonsils but in zero of two cultures from EBV-negative lines of evidence that eliminate this possibility. The level of tonsils (Fig. 7A to I). Most of the lytic staining was nuclear, infected B cells in the tonsil is ϳ1in105 (27), so to have a indicating EAD or Z protein expression, but occasional cyto- single infected cell would require the presence of ϳ105 unin- plasmic staining (VCA) was also observed (Fig. 7B and C). The fected lymphocytes, which we would readily detect in the cul- cells expressing lytic antigens were usually found in clusters, tures. Furthermore, the selective medium used in the cultures suggesting that the virus may have spread in the cultures kills all lymphocytes, including EBV-infected B cells, within a through cell-to-cell contact between epithelial cells. This is a few days. Most compelling, though, was the fact that the cells different pattern from what we observed with LMP1, which was staining positive for LMP1 and lytic proteins had the typical always detected only in single cells. This indicates that different morphology of epithelial cells. Indeed, the elimination of all cells were expressing LMP1 and the lytic antigens. These data the lymphocytes, including EBV-infected B cells, may have show that cells in primary tonsil epithelial cultures from been important in increasing our chances of detecting the rare healthy EBV-positive individuals probably sustain both latent infected epithelial cells. Where and how were these cells in- and lytic EBV infection. fected? The only possibility is through contact with infected B VOL. 78, 2004 EBV-INFECTED EPITHELIAL CELLS IN TONSILS 12621

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). cells in the explants, since the epithelial cells cannot be in- already infected at the time of explantation. It is unlikely that fected by cell-free virus. Infected B cells in the explant would we will ever be able to distinguish between these two possibil- have survived for only a day or two in the selective medium. ities, because the number of infected epithelial cells is so small Therefore, the epithelial cells would have to have become that it would be impossible to convincingly detect them on a infected before or shortly after they began migrating onto the consistent basis in the presence of all of the lymphocytes pres- tissue culture dish. Since this occurred in the presence of acy- ent when staining cross sections of intact tonsil tissue. Mech- clovir, we must conclude that these B cells were already pro- anistically, however, the two explanations both lead to the same ducing infectious virus at the time of explantation. This in turn conclusion, namely, that normal tonsil epithelium is infectible leads to the inevitable conclusion that the epithelial cells were with EBV in situ (whether it occurred in vivo or in vitro), and infected by virus-producing B cells that were already replicat- support the proposed dual tissue tropism of EBV for B and ing EBV in the tonsil. Alternatively, the epithelial cells were epithelial cells of the crypt regions of the tonsil (8, 37, 59). 12622 PEGTEL ET AL. J. VIROL.

The presence in our cultures of both latently and lytically ferent sites are heterogeneous and biologically distinct. We infected cells is fully consistent with their expected properties think, therefore, that it is important to study EBV infection in based on the behavior of EBV in the major associated epithe- the correct epithelial-tissue background. This concept is rein- lial pathologies, NPC and OHL, and other previous reports of forced by studies of EBV in B cells, where it is clear that the EBV-infected epithelial cells in vitro (10, 22, 47). Whether our pattern of viral genes expressed is dependent upon the type experiments are indicative of a role that epithelium may play in and location of the infected cell (5). It was striking that the only EBV persistence remains unresolved. It might be important to latent gene we found reproducibly in the ex vivo-infected cells note that the frequency of EBV-infected cells in the primary was the viral oncogene LMP1. At this point, it would be pre- cultures was similar to that found in B cells from the same mature to suggest that this reflects a novel form of EBV gene tonsils (ϳ0.01%). An average tonsil has a total epithelial sur- expression, i.e., LMP1 only. This is because the lower limit of 2 7 face area of 340 cm (37). This would represent ϳ5 ϫ 10 the detection sensitivity for the RT-PCR and immunohisto- tonsil epithelial cells in vivo (based on the confluency of epi- chemistry assays for the other viral latent genes is not known thelial cells in a 100-mm-diameter dish), which translates into and the infected cells are so rare. This makes a negative result ϳ 5,000 infected epithelial cells per tonsil at any given time. difficult to interpret with confidence. Nevertheless, the pres- This is comparable to the estimates of the total number of ence of LMP1 suggests that it is automatically expressed upon ϳ infected B cells in the tonsils, which is 10,000 to 50,000 (27). EBV infection of normal nasopharyngeal epithelium. Such ex- If epithelial cells do play a role in viral persistence, the pression of the oncogenic LMP1 could be the initiating event failure to infect them with cell-free virus suggests they are not that may ultimately lead to development of nasopharyngeal the target of primary infection. Their ability to be infected carcinomas. through cell-to-cell contact means that they could mediate the In vitro infection of our primary tonsil epithelial cultures release of EBV into saliva from infected B cells in the lym- required cell-cell contact, and only a small subset of cells was phoepithelium. The ready ability of epithelial cells to replicate susceptible to infection. These observations are consistent with EBV, combined with the observations that persistent infection the findings of others using different epithelial-cell types (10, is not sustainable in the absence of B cells (17), further sup- 23, 54). There is increasing evidence that viruses exhibit a high ports the idea that the primary role of epithelial cells is to degree of specificity for their targets (7). It would be interest- facilitate the release of EBV, not to maintain persistent infec- ing to see what specific characteristics the infectible cells might tion, which is presumably the function of the B-cell compart- have and if expression of particular viral-gene transcription ment. What, then, is the role of the latent infection we have programs relates to the differentiation stage of the cells. Evi- observed? If epithelial cells cannot sustain a persistent infec- dence for infection specificity in epithelial cells has recently tion, we must conclude that latent infection is transient and been shown in vitro and might depend on the expression of ␤1 either an epiphenomenon of the ability of epithelial cells to be integrin (54), which is likely to correlate with the differentia- infected or possibly a mechanism for short-term persistence, tion stage (54, 57). Our primary in vitro model with actively which might provide a more stable source of cells producing differentiating cells might prove a valuable tool to address infectious virus. these issues in the future. Virion infection of polarized epithelial cells has been reported, but only through the basolateral surface (54). It will be Early findings of EBV in oropharyngeal epithelial cells fu- necessary to discover conditions under which our primary ton- eled the notion that epithelium might play a role in viral per- sillar epithelial cells can be infected at the apical (mucosal) sistence (1, 45–47). However, subsequent reports were unable surface with cell-free virus before we can speculate that the to reproduce these findings (25, 35), casting doubt on the role epithelial-cell layer may also have a role as the first target of epithelial cells. Recent findings in culture defining an epi- tissue in primary EBV infection. thelial-cell receptor (8, 54) for the virus and suggesting a mech- There have been an ever-growing number of reports of suc- anism to explain the potential dual tissue tropism of the virus cessful infection of epithelial cells in vitro. Usually, these stud- for B cells and epithelial cells (8, 54) have reopened the issue. ies have employed established cell lines (10, 14, 16, 22, 23, 28, However, what is lacking is convincing evidence of infection in 45–47, 49, 54, 60). Attempts to infect primary nasopharyngeal, vivo. It has not been possible to detect EBV-infected epithelial ectocervical, and gastric epithelia have been described, with cells in vivo using EBER hybridization in combination with variable success. Nasopharyngeal tissue was reported to be un- B-cell and epithelial-cell-specific markers on cross sections of infectible (16); the ectocervix was reported to be infectible only tonsils and in exfoliated epithelial cells in throat washes of by oropharyngeal strains of EBV, not laboratory isolates (46); infectious-mononucleosis patients (25, 35). This approach was and the gastric epithelium was reported to be infectible in based on the assumption that during infectious mononucleosis only 3 of 21 attempts (36). We believe, therefore, that this rep- there should be a massive amount of epithelial-cell infection resents the first report of a technique to reproducibly infect that matches the levels seen in B cells, where as many as 50% representative primary cultures from tonsil epithelium. Re- of the memory cells may be infected (21). From in vitro studies, ports of viral-gene expression patterns in infected epithelium it is well known that most, if not all, B cells are infectible with have also varied. For example, EBV-infected lung, colon, and EBV; however, the in vitro experiments shown here and by primary gastric epithelial cells probably do not express LMP1 others (54) suggest that only a small fraction of tonsil epithelial upon EBV infection (10, 36, 46), but in other reports, EBV- cells are infectible with EBV. Therefore, massive infection of infected epithelial cells of naso- and oropharyngeal origin do epithelial cells may not be possible in vivo during infectious express LMP1 (10, 54). These discrepancies may in part reflect mononucleosis because, unlike B cells, most epithelial cells are technical issues but may also arise because epithelia from dif- not targets of infection. This could explain the failure to detect VOL. 78, 2004 EBV-INFECTED EPITHELIAL CELLS IN TONSILS 12623 infected epithelial cells in the tonsils of acute infectious mono- and D. A. Thorley-Lawson. 2003. Demonstration of the Burkitt’s lymphoma nucleosis patients by using histochemical techniques. Epstein-Barr virus phenotype in dividing latently infected memory cells in vivo. Proc. Natl. Acad. Sci. USA 101:239–244. Our findings support the developing model in which EBV is 21. Hochberg, D., T. Souza, M. Catalina, J. L. Sullivan, K. Luzuriaga, and D. A. able to infect healthy epithelium as part of its life cycle (8, 39, Thorley-Lawson. 2004. Acute infection with Epstein-Barr virus targets and overwhelms the peripheral memory B cell compartment with resting, latently 54, 55) and contribute to our understanding of the evolution of infected cells. J. Virol. 78:5194–5204. naso- and oropharyngeal epithelial pathologies. 22. Huang, J., H. Chen, L. Hutt-Fletcher, R. F. Ambinder, and S. D. Hayward. 2003. Lytic viral replication as a contributor to the detection of Epstein-Barr virus in breast cancer. J. Virol. 77:13267–13274. ACKNOWLEDGMENTS 23. Imai, S., J. Nishikawa, and K. Takada. 1998. Cell-to-cell contact as an efficient mode of Epstein-Barr virus infection of diverse human epithelial We thank Jim Rheinwald, Harvard Skin Disease Research Center, cells. J. Virol. 72:4371–4378. Brigham and Women’s Hospital, for advice on the culture and char- 24. Jones, P. H., and F. M. Watt. 1993. 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