The precise role of DCs in mediating HIV-1 transmission and pathogenesis at mucosal surfaces is unclear. In vitro, infection of peripheral blood mononuclear cells by HIV-1 can be strongly enhanced by including cocultured DCs (7
). While DCs are often not infected by HIV-1, virus binds to DCs efficiently and, once bound, can be retained in an infectious state for a prolonged period of time (11
). Subsequent addition of susceptible cell types results in infection by virus in trans
). The ability of DCs to bind HIV-1 and to transmit bound virus to receptor-positive cells has been linked to DC-SIGN (2
). These in vitro observations, coupled with the fact that mucosal DCs are among the first cell types encountered by HIV-1 at mucosal surfaces (10
), raise the possibility that HIV-1 interactions with DC-SIGN could impact sexual transmission.
To assess the role of DC-SIGN for virus transmission and pathogenesis, it will be important to develop reagents that specifically block virus binding to DC-SIGN. The abilities of such compounds to impact virus transmission could then be assessed in nonhuman primate models such as rhesus macaques, provided that rhesus DC-SIGN functions like human DC-SIGN and is expressed in a manner similar to that of its human homologue. We have recently shown that rhesus macaque DC-SIGN efficiently binds and transmits HIV-1, HIV-2, and SIV strains (3
). In this study, we document the expression of DC-SIGN in mucosal tissues from rhesus macaques and identify a MAb that blocks SIV interactions with DC-SIGN in vitro. Our work extends previous studies by using MAbs specific for DC-SIGN and DC-SIGNR, by studying DC-SIGN expression in macaque tissues, and by using double and triple fluorescence labeling and confocal microscopy to correlate DC-SIGN expression with expression of CD4, CCR5, and specific DC subsets. The expressions of DC-SIGN in humans and in rhesus macaques were highly similar.
In the Peyer's patches, DC-SIGN+
cells were found predominantly in the interfollicular regions, with the exception of aggregates of DC-SIGN+
cells in the subepithelial dome region. It is now clear that there is considerable variation in the localization, phenotype, and function of different DC populations in various tissues. The human DC subsets in the peripheral blood have been categorized on the basis of differential expression of myeloid (CD11c) and plasmacytoid (IL-3R) markers (reviewed in reference 21
). We show here that CD11c+
DCs are distributed in the dome and interfollicular regions and in the germinal center of the Peyer's patches, whereas the IL-3R+
plasmacytoid DCs are found exclusively in the interfollicular regions. Although all DC-SIGN+
cells expressed MHC class II molecules, we did not observe expression of DC-SIGN on IL-3R+
plasmacytoid DCs. This finding is consistent with a recent report of the lack of DC-SIGN mRNA expression by peripheral blood-derived plasmacytoid DCs (24
). Thus, plasmacytoid DCs are unlikely to participate in HIV infection of CD4+
cells in trans
, at least in a DC-SIGN-dependent manner. Further, CD11c+
DCs in the dome region were not found to express DC-SIGN. These data suggest that the DC-SIGN+
cells belong to another subset of DCs that express neither CD11c nor IL-3R within the Peyer's patches. Recently, a careful analysis of human tonsils revealed the presence of the CD11c−
DCs, which expressed MHC class II and CD68 (37
). When DC-SIGN expression on CD68+
cells was examined, we indeed observed that many of the dome region DC-SIGN+
cells coexpressed CD68 (unpublished observation). Yet, the majority of DC-SIGN+
cells in the interfollicular regions were CD68−
(unpublished observation). Future studies must address how these distinct DC subsets mediate virus transmission and immune induction during HIV-1 infection in vivo.
The epithelium covering the Peyer's patches over the dome contains M cells specialized in the uptake of luminal antigens and is known as the follicle-associated epithelium (19
). HIV-1 has been shown to gain entry through M cells in rabbit and mouse Peyer's patches, although neither species supports efficient HIV infection (1
). Thus, Peyer's patches are potentially the first sites of HIV entry during oral transmission in cases such as mother-to-child breastfeeding (9
). Further, Peyer's patches may represent sites of HIV entry following rectal exposure since rectally injected materials have been shown to be processed and presented within Peyer's patches (5
). The presence of clusters of DC-SIGN+
DC in the subepithelial dome region raises the possibility that these DCs may be the first to bind HIV-1 that enters the Peyer's patches via M cells or through tears in the mucosa. Since the dome region DC-SIGN+
DCs did not express the viral coreceptors CCR5 or CD4 at detectable levels, they are less likely to be directly infected by the virus but rather participate in infection of nearby CD4+
T cells. Aside from its HIV binding property, DC-SIGN has also been shown to interact with ICAM-3 expressed on resting T cells and mediate DC-T-cell conjugation (12
). Thus, it is also possible that DC-SIGN+
DC aggregates in the dome region acts as the gatekeeper capable of capturing, processing, and presenting HIV antigenic peptides and intact virus to CD4+
T cells within this region. Indeed, CD4+
T cells were found in close proximity to the DC-SIGN+
cells of the subepithelial dome region (Fig. ).
In the vaginal mucosa, DC-SIGN expression was not detected within the squamous epithelial layer, but it was detected on rare DCs in the subepithelial lamina propria, comparable to what has been reported for the cervix (11
). A small percentage of these cells expressed CCR5. In contrast, DC-SIGN+
DCs were abundantly distributed throughout the lamina propria immediately adjacent to the epithelium of the rectal mucosa. Moreover, DC-SIGN+
DCs that coexpressed the HIV-1 coreceptors CCR5 and CD4 formed a narrow band beneath the rectal luminal epithelium. These data are in discordance with previous studies showing that CCR5 is not expressed by mucosal DCs. Hladik and colleagues demonstrated that, unlike peripheral blood-derived DCs, cervicovaginal DCs express undetectable CCR5 but form stable conjugates with T cells which permit productive infection by HIV-1 (15
). More recently, Geijtenbeek et al. reported that CCR5 is not expressed in the rectum or uterus (11
). These apparent differences may be attributed to the sensitivities of the assays. For instance, we had to amplify the staining signal using the Tyramide amplification system in order to visualize CCR5 on these sections. Since only low levels of CCR5 are needed to support entry by many virus strains, especially if CD4 is expressed at high levels, the levels of CCR5 detected here could be relevant for virus infection (25
). Further, as discussed above, we found CCR5 expression to be restricted to the lamina propria immediately beneath the luminal epithelium in the rectum. Thus, the ability to detect CCR5+
cell population in the rectum may also depend on the tissue orientation. By the same token, it is possible that CCR5 is expressed by Peyer's patch DCs below the limit of detection by our immunofluorescence protocol, since CCR5 expression in the gut-associated lymphoid tissue has been demonstrated by a much more sensitive flow cytometry analysis (39
). Thus, future studies are needed to examine the virological relevance of the levels of the HIV coreceptors expressed by each DC subset during in vivo infection.
The examination of the relevance of mucosal DCs for the trans
infection of T cells must also take into consideration the level of DC-SIGN expressed by these cells. Not only are the tissue distributions of DC-SIGN+
cells different in the vaginal and rectal mucosae, but also the levels of DC-SIGN expression by DC in these tissues appeared to differ considerably. We have recently demonstrated that the efficiency of HIV-1 binding and transmission is strongly dependent on the level of DC-SIGN expression (26
). Although we were not able to estimate the level of DC-SIGN expressed on mucosal DCs due to the nonquantitative nature of the immunofluorescence technique, such measurements may prove useful in predicting the efficiency with which DCs in different mucosal sites can transmit HIV-1 to T cells in vivo.
Since most MAbs against DC-SIGN also detect DC-SIGNR, it was important to distinguish whether the staining was specific for DC-SIGN or DC-SIGNR. In previous studies, DC-SIGNR has been detected on endothelial cells in placenta, liver, and lymph nodes (4
). By producing a DC-SIGNR-specific MAb, we were able to show that DC-SIGNR is also expressed on about one-third of the capillary endothelial cells in the human ileum. In contrast, no DC-SIGNR-specific staining was observed in rhesus tissues. The MAb DC11, which recognizes the ectodomain of human and rhesus DC-SIGN and cross-reacts with human DC-SIGNR, did not stain rhesus endothelial cells. In fact, double labeling of rhesus Peyer's patches with MAb DC11 and DC-SIGN-specific MAb 120507 resulted in an identical staining pattern (data not shown), suggesting that MAb DC11 detects only DC-SIGN in rhesus tissues. Thus, either the rhesus DC-SIGNR homologue is distinct in the regions recognized by MAb 120604 and MAb DC11, it is not expressed in the tissues we examined, or no homologue for DC-SIGNR exists in rhesus macaques.
If DC-SIGN plays a role in virus transmission, then the accessibility of DC-SIGN+
DCs within different mucosal exposure sites would likely influence the efficiency of this process. During vaginal exposure, virus must somehow cross the epithelial layer to reach DC-SIGN+
DCs in the subepithelial lamina propria. It is interesting that progesterone treatment, which results in thinning of the vaginal epithelium, has been shown to contribute to a higher incidence of vaginal SIV transmission in rhesus macaques (22
). Hormonal influences and microbial flora within the vaginal mucosa that result in thinning of the epithelium may potentially enhance the ability of HIV to gain access to the DC-SIGN+
cells in the lamina propria. In contrast, DC-SIGN+
DCs in the rectal mucosa are separated from the lumen by only a single columnar epithelium, which should allow virus access to DC-SIGN+
DCs more easily. This hypothesis is corroborated by the fact that the HIV-1 transmission risk is greater in anal intercourse than in vaginal coitus in women (40
In summary, DCs expressing DC-SIGN were distributed similarly in the mucosal surfaces of humans and rhesus macaques. The physical barriers that exist between the lumen and the closest DC-SIGN+ DCs were greatest in the vaginal mucosa and least in the rectum. An intriguing finding, however, is that the DC-SIGN+ DCs located near the lumen of the rectum were also positive for CD4 and CCR5. These DCs may not only ferry HIV to the draining lymph nodes but also become infected within the rectal mucosa and form a local viral factory in DC-T-cell conjugates. Further understanding of this viral adhesion molecule may provide insight into its potential use in novel preventative microbicidal agents against HIV transmission.