By using cytospin slides of HLA-ECS, our analysis focused on the antigen-presenting cells of the genital tract, including immature CD1a+
LC and mature P55+
DC. Thus, we were able to definitively demonstrate that DC in general, and LC in particular, are target cells for primary SIV infection in the vaginal epithelium and lamina propria of rhesus macaques. These infected DC were detected in the first 18 to 24 h after vaginal SIV exposure. However, even in the HLA-ECS cytospin slides, the two types of DCs we identified accounted for only 50 to 90% of the SIV RNA+
cells in the samples. Thus, a considerable number of cells other than DCs are infected in the first 24 h of SIV exposure. Aside from a single animal in which SIV-infected CD3+
T cells were identified in the vaginal lamina propria HLA-ECS, we were unable to immunophenotype the SIV-infected cells that were not LC or DC in the limited sample material available. The unidentified SIV RNA+
cells consisted of two morphologic types, small, round cells with scant cytoplasm and round nuclei or medium-sized, irregularly round to oval cells with abundant cytoplasm and kidney-shaped nuclei. Based on morphologic criteria, these other cell types are lymphocytes and macrophages, respectively. The finding of SIV RNA+
macrophages in the iliac lymph node at 18 h PI (Fig. E) also supports this interpretation. Thus, we conclude that the initial target cells for SIV during vaginal transmission include large numbers of LC, DC, T lymphocytes, and macrophages. It has been shown that vaginal LC take up antigen from the vaginal lumen and then migrate to the T-cell-rich paracortex of the draining lymph nodes (27
). The data presented support the conclusion that intraepithelial DC are critical initial target cells after intravaginal SIVmac251 inoculation. DC may play a similar role in heterosexual transmission of HIV to women.
Our ability to document DC infection immediately after mucosal SIV exposure contrasts with the results of several other groups (32
). The different results can be explained largely on the basis of methodological differences in the studies. The kinetics of DC antigen uptake from the vagina and subsequent migration to draining lymph node (27
) led us to focus our analysis on events occurring in the genital mucosa within 24 h of SIV exposure. The ISH assay used in this study was not able to detect SIV in T-cell lines until 12 h after in vitro infection. Thus, the time points after inoculation that we chose to examine were an attempt to balance the need to allow viral RNA expression to reach detectable levels and the need to obtain the tissue samples before substantial DC migration occurred. The cell sorting strategy, designed to enrich our samples for DC, also maximized the probability that we would detect DC infection. In addition, because cytoplasmic RNA is more accessible to hybridization in cytospin slides than in formalin-fixed, paraffin-embedded tissues, we were able to sensitively detect infected DC. Another advantage of the cytospin preparations is that immunophenotypic characterization of infected cells does not require antigen retrieval and a broader range of antibodies is available to detect cell surface markers. Our ISH protocol uses seven riboprobes, some of which detect expression of regulatory genes that are expressed relatively early in the viral life cycle. The sensitivity of the standard ISH NBT/BCIP assay (without Tyramide amplification) was confirmed by our ability to detect SIV RNA+
cells 12 h after in vitro infection of T-cell lines or PBMC (data not shown).
The number of SIV RNA+ cells in the vaginal mucosa can be estimated by using the frequency of SIV RNA+ cells in the formalin-fixed histologic sections of vagina. On average, we detected one SIV RNA+ cell in each 6-μm-thick section of vaginal mucosa. Once opened along the longitudinal axis, the rhesus macaque vagina is approximately 4 by 7 cm. At least 11,600 histologic tissue sections (6 μm thick) can be produced from a tissue sample of that size. Assuming that the frequency of one SIV RNA+ cell per section of vagina is accurate, then approximately 10,000 cells in the vaginal mucosa, mostly DC and LC, became infected with SIV within 18 h of intravaginal inoculation with 105 TCID50 of SIVmac251. This may be an underestimate, considering that an infected cell must contain at least 10 RNA copies for detection by ISH and that transcriptionally inactive provirus cannot be detected.
A detailed discussion of the relevance of the SIV-rhesus macaque model to heterosexual HIV transmission is beyond the scope of this study, and a number of reviews are available (15
). Briefly, it is widely accepted that the HIV variants transmitted by sexual contact are macrophage-tropic and use CCR5 as a coreceptor (reviewed in reference 16
). SIVmac251, used in our studies, replicates well in primary macrophages and uses CCR5 as a coreceptor (3
). The inoculum contains high-titer virus (105
RNA copies/ml). We have shown that, while inoculation with a low-titer inoculum can produce systemic infection in rhesus macaques, the efficiency of transmission with a particular virus stock is directly related to the titer of infectious virus inoculum (20
). A similar relationship between the virus load in an HIV-infected person and transmission to an uninfected partner is well established (5
). Use of the high-titer SIV inoculum increases the probability of interactions between infectious virions and susceptible target cells in the genital tract, but it is unlikely to alter the basic biology of the virus-target cell relationship. In fact, the frequencies and types of virus-infected cells in the genital tracts of chronically SIV-infected female rhesus macaques and HIV-infected women are similar (15
). Thus, in both species, lentivirus-infected macrophages, T cells, and DC can be routinely detected in the female genital tract during the chronic stage of the infection (7
). Studies using human tissues collected in the first few hours after HIV exposure can never be conducted to verify the findings reported here. However, the similarities between tissue-based studies in chronic SIV and HIV infection in the female genital tract suggest that the findings in the SIV model are relevant to HIV sexual transmission. It is worth noting that in chronic SIV infection, there may be regional differences in the types of cells that are infected at different mucosal surfaces. Numerous SIV-infected DC are found in the genital tract of an animal, but they are difficult to detect in the tonsils of the same animal (6
). Thus, other mucosal tissues, such as tonsils, cannot be used as surrogates for studying genital tract HIV infection. In fact, these regional differences may exist between the endocervix and the rest of the cervicovaginal mucosa, and findings in one tissue cannot be extrapolated to the other.
The results of the experiments described here are consistent with the hypothesis (23
; L. R. Braathen, G. Ramirez, R. O. Kunze, and H. Gelderblom, Letter, Lancet 2:
1094, 1987) that intraepithelial DC are the initial target cells of HIV infection in the genital tract. We also provide evidence that these infected DC then migrate to draining lymph nodes, where the infection is passed to CD4+
T lymphocytes that disseminate the virus systemically as they recirculate through the body. It was recently demonstrated that following intravaginal inoculation of mice with HIV, DC take up and transport virus to genital lymph nodes in less than 24 h (14
). Thus, in vivo experiments in both mice and monkeys now support the hypothesis (23
; Braathen, et al., Letter) that DC play a critical role in disseminating HIV from the genital tract to lymphoid tissues in the first 24 h after virus exposure.
The results also suggest that a second pathway of dissemination may be involved in HIV sexual transmission. We found SIV RNA+
T lymphocytes in the genital tract of one animal at 18 h PI. The ISH technique used for these studies could not detect SIV RNA expression until 12 h after in vitro infection of T-cell lines; thus, it is unlikely that the T-cell infection represents passage of the infection from infected DC to the T cells. Apparently the T cells were directly, and rapidly, infected by the inoculum. The mechanism of T-cell infection is unclear, as the vaginal epithelium provides a barrier to the entry of water-soluble dyes and presumably larger particles, such as lentiviruses, from the vaginal lumen into the mucosa (10
). A few CD4+
T cells are present in the cervicovaginal epithelium of rhesus macaques (21
), and these cells could be directly infected if they entered the superficial layers of the epithelium. It is also possible that there were breaks in the vaginal epithelium which provided the virus direct access to CD4+
T cells in the lamina propria, but we did not see such features in the histologic slides examined. The early infection of T cells after mucosal inoculation is consistent with the results of other SIV mucosal transmission studies (33
) and may explain the presence of the SIV provirus detected in lymphoid tissues beyond the lymph nodes that drain the genital tract by PCR. If T cells were directly infected in the genital tract, then they could enter the peripheral vasculature and recirculate widely, disseminating the infection. Further study is required to determine the relative significance of these two pathways of viral dissemination from the genital tract.
We have documented the rapid penetration of SIV into the genital mucosa, infection of intraepithelial DC, and dissemination of SIV-infected cells to the draining lymph node within hours of vaginal exposure to the virus. These findings may have practical implications for developing strategies to block HIV sexual transmission. If our findings related to vaginal SIV transmission accurately reflect HIV biology, then HIV infects DC and begins to disseminate very rapidly after sexual contact. It would appear that, in order to stop systemic spread of HIV infection after genital exposure, a vaccine will need to elicit potent immunologic memory cell populations that rapidly expand in response to the presence of HIV recall antigens in the genital tract.