Unprotected sexual intercourse is the most common mode of HIV transmission world-wide [1,2]. Many biological factors have been implicated in influencing the rate of transmission from individual exposures, including virus strain and inoculum in semen or vaginal fluid, concomitant ulcerative anogenital infection, traumatic intercourse, menstrual cycle, use of oral contraceptives, and genetic traits [3–6]. Although sexual transmission of HIV is a relatively common occurrence, the early cellular and molecular events that take place during this biological process are poorly understood. Much of this lack of knowledge can be attributed to the lack of good animal and ex vivo tissue culture models.
Sexual transmission of HIV involves transport of virus from mucosal surfaces to CD4 T cells within regional lymph nodes. To model this process, cell culture systems for HIV transmission have focused on Langerhans cells (LC), members of the dendritic cell family, because they exhibit all of the following characteristics: (i) location within mucosal epithelium at sites of HIV exposure [7,8]; (ii) expression of CD4, CCR5, and CXCR4 capable of supporting HIV and simian immunodeficiency virus (SIV) infection both in vivo and in vitro[9–17]; (iii) the ability to emigrate to paracortical T cell-rich areas of regional lymph nodes following contact with virus or other antigens [7,8]; and (iv) the ability to induce clustering, potent activation, and efficient transmission of HIV in LC–T cell cocultures [9–11,18,19]. No other potential initial target cell type for HIV, including T cells, monocytes/macrophages, epithelial cells, and blood-derived dendritic cells, possess all of these salient features. Mucosal epithelial cells are CD4 and HIV coreceptor negative and there is little evidence for HIV or SIV infection of these cells [16,17,20]. Intraepithelial T cells and macrophages, other potential initial targets for HIV, express CD4 and HIV coreceptors and are certainly susceptible to infection by virus, but they do not have the specific migratory potential of LC. In addition, when compared on a cell-per-cell basis, macrophages do not induce nearly as potent activation of CD4 T cells as do dendritic cells [7,8]. Thus, it would be hard to invoke a primary role for these cells in transporting virus to regional lymph node and transmitting infection to activated CD4 T cells.
In this report, a novel model for LC-mediated viral infection of lymphoid tissue, one early event involved in primary HIV infection, is described. The strength of this model system relies on the use of human epithelium-derived LC and human lymphoid tissue, which are particularly relevant cells and tissues involved in sexual transmission of HIV.
Materials and methods
Preparation of human epidermal LC
The Institutional Review Board of the National Cancer Institute approved acquisition of human skin and informed consent was obtained from all healthy volunteers. As described , blisters were induced by vacuum suction and heat on normal-appearing skin; blister roofs (i.e., epidermal sheets) were then removed with sterile scissors, washed in sterile phosphate- buffered saline (Biofluids, Inc., Rockville, Maryland, USA), and trypsinized to make epidermal cell suspensions. For some experiments, LC were depleted from epidermal cell suspensions using anti-CD1a monoclonal antibodies (mAb) and immunomagnetic bead separation as described previously . LC depletion efficiency was confirmed by staining depleted and undepleted cell populations with anti-HLA-DR mAb, which specifically labelled LC within epidermal cell suspensions obtained from healthy human skin . LC-depleted or non-depleted epidermal cells were then resuspended at 1 × 106 cells/ml in RPMI 1640 (Gibco, Grand Island, NY) supplemented with heat-inactivated 10% (v/v) human AB serum (Sigma Chemical Co., St. Louis, Missouri, USA), 100 U/ml penicillin (Gibco), 100 μg/ml streptomycin (Gibco), 2 mM l-glutamine (Gibco), and 5 × 105 M 2-ME (Sigma) (complete medium) and cultured overnight at 37°C in a humidified 5% CO2 environment. Freshly prepared epidermal cell suspensions consisted of keratinocytes (approx. 95% of all cells), LC (approximately 2–3%), melanocytes (approximately 1–2%), and no resident T cells , whereas approximately 10% of 1-day-cultured epidermal cell suspensions consisted of LC [14,15] because of preferential death of cultured keratinocytes (compared with cultured LC). As controls, cultures of primary human keratinocytes completely devoid of LC were purchased from Clonetics Corp. (San Diego, California, USA) and grown according to the supplier's instructions.
Preparation of human lymphoid tissue ex vivo
As described in detail previously [23,24], human tonsillar tissue removed during therapeutic tonsillectomy and not required for clinical purposes was placed in phosphate-buffered saline and delivered to the laboratory within 5 h of excision. The tonsils were washed thoroughly with medium containing antibiotics and then sectioned into 2–3 mm blocks. These tissue blocks (histocultures) were placed on top of collagen sponge gels in culture medium at the air–liquid interface and incubated overnight at 37°C in a humidified 5% CO2 environment, prior to exposure to epidermal cells and/or HIV.
Viral strains and infection assays
Purified, pelleted, and titrated HIVBa−L(R5) and HIVIIIB (X4) were purchased from Advanced Biotechnologies Inc. (Columbia, Maryland, USA). The HIV laboratory-adapted strains HIVSF162 (R5) and HIVLAV.04 (X4), as well as the HIV primary isolates #91US056 (R5) and #93US151 (X4), were obtained from the AIDS Research and Reagent Program, Division of AIDS, NIAID, NIH (Bethesda, Maryland, USA). Predominant coreceptor usage for each strain was reconfirmed using a method described previously by others .
For infection of LC, 1-day cultured epidermal cell suspensions were resuspended at 1 × 106 cells/ml in complete medium and incubated with various isolates and dilutions of HIV for 4 h at 37°C in a humidified 5% CO2 environment. Cells were then harvested and washed three times in 50 ml wash media. For all HIV infection experiments, supernatants from the final wash were collected and placed directly onto tonsil histocultures as a control for residual cell-free viral infection. After washing, 2 × 105 HIV-exposed epidermal cells (approximately 2 × 104 LC) were then applied to each tonsil tissue block (approximately 2.4 × 105 CD4 T cells/block, corresponding to a CD4 T cell : LC ratio of approximately 10). For some experiments, 1-day cultured LC-depleted epidermal cell suspensions or primary keratinocytes cultures were exposed to HIV and placed onto tonsil histocultures in a manner identical to that described for non-depleted cell suspensions. For positive controls of tissue susceptibility of R5 and X4-HIV infection, cell-free HIV (0.3–1.2 ng p24 content) was applied directly to the surface of each tissue block. Each type of experiment was performed at least three times. Productive HIV infection in histocultures was assessed by measuring secreted p24 protein in culture media by ELISA (Cellular Products, Buffalo, New York and AIDS Vaccine Program, NCI, Frederick, Maryland, USA). Specifically, the concentration of p24 accumulated in 3 ml of culture medium bathing 10 tissue blocks during the 2–3 days between successive media changes was used a measure of HIV replication.
Results and discussion
Here, human LC are shown for the first time to be the major epidermal cell type responsible for transmitting HIV infection to human lymphoid tissue in a novel ex vivo model. Epidermal cell suspensions containing LC or epidermal cells suspensions depleted of LC were exposed to HIV, thoroughly washed, applied to tonsil surfaces, and monitored for evidence of HIV replication. The efficiency of the LC depletion procedure was first reconfirmed; immunomagnetic bead-treated epidermal cell suspensions always showed < 0.05% remaining LC within epidermal cell suspensions by mAb staining and flow cytometry (Fig. 1a). In some experiments, LC-depleted epidermal suspensions exposed to HIV were not able to transmit infection to tonsils, whereas in other experiments very low levels of infection were observed (Fig. 1b, c). These low levels of infection were attributed either to infection of rare LC that had survived the depletion procedure or to HIV that was carried or trapped on surfaces of keratinocytes (i.e., epithelial cells). To investigate this latter possibility, cultures of primary human keratinocytes completely devoid of LC were exposed to HIV as described above and then applied to surfaces of tonsils. Surprisingly, these cells were also able to transmit low levels of infection to tonsil histocultures (Fig. 1b, c). As these cells do not express CD4 and HIV coreceptors [14,15], this keratinocyte `transmission' was attributed to carry over of virions on the surfaces of cells that persisted during the washing procedure, as it is known that small amounts of cell-free HIV and SIV can efficiently infect tonsil tissue [23,24,26].
LC as shown here were capable of transmitting infection to human lymphoid tissue using a variety of both laboratory-adapted and primary HIV isolates (with both R5- and X4-tropism) (Fig. 2). This is consistent with findings previously reported showing that cultured human epidermal LC express CD4, CCR5, and CXCR4 and can be infected by both R5- and X4-HIV [13–15]. Thus, because this model uses activated LC (that have increased CXCR4 surface expression compared with freshly prepared LC ) it cannot be used for evaluation of the role of LC in the selective sexual transmission of R5-HIV strains.
One difficulty in firmly establishing a role for LC in initial HIV infection has been the interpretation of results obtained in SIV-macaque studies. Atraumatic vaginal inoculation of SIV in female macaques leads to systemic SIV infection [20,27–29]. When mucosal tissues were examined 1–2 days following inoculation, no evidence of intraepithelial LC infection was documented [20,26,29]. However, HIV-infected LC are expected to emigrate within a few hours of HIV exposure, so that tissue from early time points (e.g., at 2, 4, and 6 h) needs to be examined. Secondly, assays that detect only productive HIV infection within tissue, e.g., p24 staining, would be expected to show negative staining in HIV-exposed activated LC. This assumption is based on the in vitro findings that immature dendritic cells can be productively infected with HIV, but that initiation of the activation process leads to non- productive latent HIV infection within mature dendritic cells [11,13,19]. Thus, proving a definitive role for LC in initial HIV infection will require assessment of latently infected cells at very early time points following exposure.
Using a novel ex vivo model, human LC are shown here for the first time to be the epidermal cell type responsible for the majority of HIV infection that is subsequently carried into and established within human lymphoid tissue. The strength of this model system relies on the use of human epithelium-derived LC and human lymphoid tissue. Both R5- and X4-HIV could be transmitted using this model, consistent with previously reported HIV coreceptor expression on activated LC [13–15] and lymphoid T cells . Importantly, this system could prove useful in further understanding LC trafficking and other early biological events involved in primary HIV infection.
The authors thank I. Tokar for assisting with the healthy volunteers, H. Schaefer for preparing the figures, and T. Kawamura, S.S. Cohen, D.I. Cohen, M. Qalbani, and E.A. Aquilino for technical assistance.
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