Accumulating evidence indicates that infection of circulating monocytes by herpesviruses, including human cytomegalovirus, Kaposi's sarcoma-associated herpesvirus, and herpesvirus 6, may play a critical role in establishing a reservoir for latent infection in the blood compartment (2
). EBV infection of monocytes, macrophages, and dendritic cells has been observed in vitro and in vivo (15
). Detection of EBV-infected macrophages in healthy asymptomatic individuals (36
) suggests that, like B lymphocytes, circulating monocytes and tissue macrophages may serve as a reservoir for EBV infection. We also found EBV-positive circulating monocytes in both HIV-negative and HIV-positive individuals. The presence of EBV DNA in circulating monocytes has been shown recently by Schlitt et al. (33
). Since circulating monocytes can migrate to tissue sites (17
), including oral mucosa (5
), EBV-infected monocytes may serve as a vehicle for virus transmission between the blood compartment and oral epithelium. Detection of similar EBV strains (29
) in WBC and HL and the absence of reinfection of epithelium by WBC of EBV-eradicated bone marrow transplants (11
) suggest that EBV infection of oral epithelium may occur from the blood compartment.
Consistent with this hypothesis, our data from oral tissue explants infected ex vivo with cell-free and cell-associated EBV showed that EBV-infected macrophage and dendritic cell precursors from the lamina propria migrate into the mucosal epithelium and infect oral keratinocytes within the spinosum layer. Expression of tight junction proteins in EBV-infected intraepithelial macrophages/LC indicated their migrating status (6
). Reduction of EBV infection of oral keratinocytes by inhibiting migration of EBV-infected monocytes to the mucosal epithelium using CCR2 and MCP-1 antibodies indicated that EBV-infected monocytes may play a key role in EBV dissemination within the oral epithelium. Also consistent with infection of epithelium from the blood compartment was our observation that the mucosal surface of oral epithelium was resistant to EBV infection, as well as our earlier findings that EBV did not enter from the apical surface of polarized oral keratinocytes (42
). This mechanism of spread of EBV to epithelium may also be occurring in cutaneous T-cell lymphoma, in which both keratinocytes (8
) and intraepithelial LC of skin T-cell lymphoma were shown to be positive for EBV (19
In comparison with EBV-positive monocytes, migration of EBV-infected B lymphocytes into mucosal epithelium was rare and inefficient. However, we cannot completely exclude a role for EBV-infected B lymphocytes in virus spread within the oral epithelium. Rare EBV-infected intraepithelial B and T lymphocytes have been described previously in nasopharyngeal mucosa (41
). EBV replication in vivo occurs in terminally differentiated plasma B lymphocytes (21
), and these cells may migrate into mucosal epithelium and play a role in virus dissemination within the mucosal epithelium. We did not detect EBV-infected submucosal or intraepithelial CD138+
plasma B lymphocytes in any of the tissue explants that were cocultivated with EBV-infected B lymphocytes. This could reflect the lack of essential factors for terminal differentiation of B lymphocytes in our ex vivo explant system, and if so, our system may not be sufficient to fully evaluate the role of B lymphocytes in EBV spread within the oral epithelium. However, the lack of EBV-infected CD19+
cells in HL strongly suggests that the role of B lymphocytes in EBV spreading to oral epithelium is limited at best. Furthermore, it has been shown that EBV infection in B lymphocytes inhibits CXCR4 expression on the B-lymphocyte surface (9
), leading to reduced migration of EBV-infected B lymphocytes toward stromal cell-derived factor 1 in vitro (9
). Stromal cell-derived factor 1 expression has been detected in tonsil epithelium (4
), and we also detected its expression in tongue and buccal mucosal epithelium (data not shown). Thus, the absence or low level of EBV-infected B-lymphocyte migration into oral mucosal epithelium may be due to inhibition of CXCR4 expression, consistent with a lack of a significant role for B lymphocytes in EBV dissemination within the oral epithelium.
Detection of EBV infection in keratinocytes in only 1 of 20 tongue explants infected with cell-free EBV indicated that cell-free EBV infection of keratinocytes was very inefficient. In contrast, EBV infection of keratinocytes of tongue explants cocultivated with EBV-infected monocytes was highly efficient, indicating that dissemination of EBV to keratinocytes depends on the manner of EBV presentation to these cells, the number of EBV-infected monocytes, and the duration of their exposure to the explants. A small number of EBV-infected keratinocytes (approximately five cells/section) was detected in three oral explants cocultivated with EBV-infected B lymphocytes. In these explants EBV-infected intraepithelial B lymphocytes were not found, indicating that these B lymphocytes were not able to spread virus within the oral epithelium. In contrast, EBV-infected submucosal and intraepithelial macrophages/LC were detected in these explants. While it has been shown elsewhere that B lymphocytes may transfer membrane-bound EBV virions into epithelial cells (35
), our data show that EBV may spread from B lymphocytes and into monocytes. The presence of EBV-infected macrophages/LC in these explants suggests that a likelier scenario is that EBV-infected B lymphocytes in the lamina propria transferred EBV to macrophages/LC, and these EBV-infected macrophages/LC then migrated into the epithelium and spread virus the keratinocytes (Fig. ). However, we cannot exclude the possibility that the monocytes could be infected with EBV in the circulating blood compartment from B lymphocytes. If this were to occur, these EBV-infected circulating monocytes subsequently might migrate into mucosal epithelium, differentiate into macrophages and LC, and disseminate EBV within the oral epithelium (Fig. ).
FIG. 9. Model of EBV infection in stratified oral mucosal epithelium. In immunocompromised individuals, particularly those with HIV-mediated immunodeficiency, EBV replication may reactivate in latently infected B lymphocytes, and infectious progeny virions from (more ...)
Like the explants infected with EBV ex vivo, analysis of HL tissues also showed the presence of EBV-infected submucosal and intraepithelial macrophages and LC. Individuals with HIV infection have both higher numbers of circulating EBV-infected monocytes and higher EBV copy numbers per cell than HIV-negative individuals. While we cannot exclude possible detection of EBV in the monocyte populations due to B-lymphocyte contamination, the level of B-lymphocyte contamination of the monocyte population was low at 0.1 to 0.5%, and this small number of cells could not account for the high EBV copy numbers in the monocyte population. EBV infection of circulating monocytes could be due to EBV reactivation in B lymphocytes (23
) in the setting of HIV-associated immunosuppression. Migration of EBV-infected monocytes into oral mucosal epithelium may then spread EBV infection to keratinocytes within the epithelium and initiate development of HL.
It has been shown elsewhere that EBV replication is activated in the tongue in the early stages of HIV infection, well before HL becomes clinically apparent (3
). The depletion of EBV-positive intraepithelial macrophages and LC in HL lesions (7
) also suggests that intensive migration of EBV-infected monocytes into oral epithelium might occur at an early stage of HL development. Reduction of EBV-infected intraepithelial macrophages/LC at 7 days post-infection of oral explants suggested that EBV infection might cause the death of these cells. Alternatively, detection of CD86 in EBV-infected intraepithelial macrophages/LC suggested that these activated immune cells may migrate in the opposite direction, i.e., from the epithelium toward the lamina propria (1
). Thus, EBV infection of oral mucosal macrophages/LC may be involved in depletion of macrophages/LC in HL epithelium.
In summary, our findings show that intraepithelial EBV-infected monocytes/macrophages/LC migrate from the submucosa into the epithelium and disseminate EBV infection within the spinosum/granulosum layers of oral epithelium. These data suggest that EBV infection of oral epithelium via intraepithelial monocytes/macrophages/LC may play a key role in development of the HL lesion during immunosuppression, particularly at the early stages of lesion development before macrophages/LC cells become depleted. This may also be the major mechanism by which low-level EBV infection of oral epithelium occurs in an ongoing basis in healthy individuals.