Objectives: To create a novel ex vivo model for early biologic events involved in sexual transmission of HIV and to demonstrate that Langerhans cells (LC), the purported initial mucosal target cells for HIV, play a critical role in this process.
Methods: Epidermal cells containing LC were isolated from normal-appearing skin of healthy volunteers and exposed to a panel of primary and laboratory-adapted R5- and X4-HIV isolates, washed and applied to the surfaces of allogeneic tonsil tissue blocks. Viral replication was followed by measuring HIV p24 protein in culture supernatants by ELISA.
Results: Both R5- and X4-HIV isolates could be transmitted by LC and established high levels of infection in lymphoid tissue (p24 > 10 ng/ml). Depletion of LC within epidermal cell suspensions abrogated the ability of HIV-exposed suspensions to transmit virus to tonsil histocultures.
Conclusions: Using a novel ex vivo model, human LC are shown for the first time to be the major epidermal cell type that is involved in transmission of HIV infection to human lymphoid tissue. Importantly, this system could prove useful in further understanding LC trafficking and other early biological events involved in primary HIV infection.
From the Dermatology Branch, National Cancer Institute and the aLaboratory of Molecular and Cellular Biophysics, National Institute of Child Health and Human Development, Bethesda, Maryland, USA.
Note: A. Blauvelt and S. Glushakova contributed equally to this work.
Requests for reprints to: A. Blauvelt, Dermatology Branch, National Cancer Institute, Building 10/Room 12N238, 10 Center Dr. MSC 1908, Bethesda, MD 20892-1908.
Received: 12 December 1999; accepted: 7 January 2000.
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.
1. Royce RA, Sena A, Cates W, Cohen MS. Sexual transmission of HIV. N Engl J Med 1997, 336: 1072–1078.
2. Kahn JO, Walker BD. Acute human immunodeficiency virus type 1 infection. N Engl J Med 1998, 339: 33–39.
3. Cohen MS. Sexually transmitted diseases enhance HIV transmission: no longer a hypothesis. Lancet 1998, 351 (suppl 3): 5–7.
4. Vernazza PL, Eron JJ, Fiscus SA, Cohen MS. Sexual transmission of HIV: infectiousness and prevention. AIDS 1999, 13: 155–166.
5. Cohen OJ, Fauci AS. Host factors that affect sexual transmission of HIV. Int J Infect Dis 1998, 2: 182–185.
6. Miller CJ. Host and viral factors influencing heterosexual HIV transmission. Rev Reprod 1998, 3: 42–51.
7. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998, 392: 245–252.
8. Jakob T, Udey MC. Epidermal Langerhans cells: from neurons to nature's adjuvants. Adv Dermatol 1999, 14: 209–259.
9. Weissman D, Fauci AS. Role of dendritic cells in immunopathogenesis of human immunodeficiency virus infection. Clin Microbiol Rev 1997, 10: 358–367.
10. Zoeteweij JP, Blauvelt A. HIV-dendritic cell interactions promote efficient viral infection of T cells. J Biomed Sci 1998, 5: 253–259.
11. Pope M, Betjes MGH, Romani N. et al
. Conjugates of dendritic cells and memory T lymphocytes from skin facilitate productive infection with HIV-1. Cell 1994, 78: 389–398.
12. Tschachler E, Groh V, Popovic M. et al
. Epidermal Langerhans cells-a target for HTLV-III/LAV infection. J Invest Dermatol 1987, 88: 233–237.
13. Granelli-Piperno A, Moser B, Pope M. et al
. Efficient interaction of HIV-1 with purified dendritic cells via multiple chemokine coreceptors. J Exp Med 1996, 184: 2433–2438.
14. Zaitseva M, Blauvelt A, Lee S. et al
. Expression and function of CCR5 and CXCR4 on human Langerhans cells and macrophages: implications for HIV primary infection. Nature Med 1997, 3: 1369–1375.
15. Zoeteweij JP, Golding H, Mostowski H, Blauvelt A. Cutting edge: cytokines regulate expression and function of the HIV coreceptor CXCR4 on human mature dendritic cells. J Immunol 1998, 161: 3219–3223.
16. Hu J, Pope M, Brown C, O'Doherty U, Miller CJ. Immunophenotypic characterization of simian immunodeficiency virus-infected dendritic cells in cervix, vagina, and draining lymph nodes of rhesus monkeys. Lab Invest 1998, 78: 435–451.
17. Zhang L, He T, Talal A, Wang G, Frankel SS, Ho DD. In vivo distribution of the human immunodeficiency virus/simian immunodeficiency virus coreceptors: CXCR4, CCR3, and CCR5. J Virol 1998, 72: 5035–5045.
18. Cameron PU, Freudenthal PS, Barker JM, Gezelter S, Inaba K, Steinman RM. Dendritic cells exposed to human immunodeficiency virus type-1 transmit a vigorous cytopathic infection to CD4+ cells. Science 1992, 257: 383–387.
19. Blauvelt A, Asada H, Saville MW. et al
. Productive infection of dendritic cells by HIV-1 and their ability to capture virus are mediated through separate patbility to capture virus are mediated through separate pathways. J Clin Invest 1997, 100: 2043–2053.
20. Zhang ZQ, Schuler T, Zupancic M. et al
. Sexual transmission and propagation of SIV and HIV in resting and activated CD4(+) T cells. Science 1999, 286: 1353–1357.
21. Blauvelt A, Clerici M, Lucey DR. et al
. Functional studies of epidermal Langerhans cells and blood monocytes in HIV-infected persons. J Immunol 1995, 154: 3506–3515.
22. Blauvelt A, Asada H, Klaus-Kovtun V, Altman DJ, Lucey DR, Katz SI. Interleukin-15 mRNA is expressed by human keratinocytes, Langerhans cells, and blood-derived dendritic cells and is downregulated by ultraviolet B radiation. J Invest Dermatol 1996, 106: 1047–1052.
23. Glushakova S, Baibakov B, Margolis LM, Zimmerberg J. Infection of human tonsil histocultures: a model for HIV pathogenesis. Nature Med 1995, 1: 1320–1322.
24. Grivel JC, Margolis LB. CCR5- and CXCR4-tropic HIV-1 are equally cytopathic for their T-cell targets in human lymphoid tissue. Nature Med 1999, 5: 344–346.
25. Deng HK, Unutmaz D, Kewal-Ramani VN, Littman DR. Expression cloning of new receptors used by simian and human immunodeficiency viruses. Nature 1997, 388: 296–300.
26. Stahl-Hennig C, Steinman RM, Tenner-Racz K. et al
. Rapid infection of oral mucosal-associated lymphoid tissue with simian immunodeficiency virus. Science 1999, 285: 1261–1265.
27. Miller CJ, Alexander NJ, Sutjipto S. et al
. Genital mucosal transmission of simian immunodeficiency virus: animal model for heterosexual transmission of human immunodeficiency virus. J Virol 1989, 63: 4277–4284.
28. Sodora DL, Gettie A, Miller CJ, Marx PA. Vaginal transmission of SIV: assessing infectivity and hormonal influences in macaques inoculated with cell-free and cell-associated viral stocks. AIDS Res Hum Retroviruses 1998, 14 (suppl 1): S119–S123.
29. Spira AI, Marx PA, Patterson BK. et al
. Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques. J Exp Med 1996, 183: 215–225.