Ex Vivo Modeling of Oral HIV Transmission in Human Palatine Tonsil
Departments of Microbiology (DMM,Z-QZ,PJS) and Medicine (TWS), University of Minnesota, Minneapolis, Minnesota
Correspondence to: Peter Southern, Department of Microbiology, MMC 196, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455. E-mail: peter{at}mail.ahc.umn.edu
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Summary |
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Key Words: palatine tonsil human organ culture primary epithelial cells HIV infection lymphocyte activation confocal microscopy time-lapse imaging
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Introduction |
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Oral transmission of HIV is believed to occur following the introduction of cell-free virions or HIV-infected cells into the oral cavity. Both milk and semen are recognized as potential sources of HIV infectivity for oral transmission by nursing and by receptive oral intercourse, respectively (Crittenden et al. 1992; Dunn et al. 1992
; Black 1996
; Vernazza et al. 1996
; Southern 1998
; Semba et al. 1999
). Additional evidence supporting a direct connection between exclusive oral exposure and subsequent establishment of disseminated virus infection has been derived from atraumatic oral infections in the SIV/rhesus macaque model system (Baba et al. 1996
; Stahl-Hennig et al. 1999
). SIV-infected cells have been detected in macaque palatine tonsil, but many details relating to virus exposure and the initiation of primary infection have not been fully resolved.
The palatine tonsil is one of several secondary lymphoid tissues situated in the human pharynx that constitutes part of the mucosa-associated lymphoid tissue (MALT), reviewed in (Perry 1994; Neutra et al. 1996
; Perry and Whyte 1998
). The bulk of the external surface of the palatine tonsil is protected by a stratified squamous epithelium, but there are periodic invaginations (tonsillar crypts) that increase the total surface area of tonsil
6-fold (Perry and Whyte 1998
). The crypts are lined by a reticulated epithelium that is populated predominantly by epithelial cells and leukocytes. The specialized composition of cryptal reticulated epithelium is connected with antigen sampling, and functional parallels have been drawn between palatine tonsil and Peyer's patches in the intestine (Neutra et al. 1996
). There have been many suggestions that tonsillar crypts may be exploited as a portal of entry by both bacteria and viruses in the oral cavity (Mbopi-Keou et al. 2002
), and we have therefore designed a series of experimental systems to analyze the events surrounding exposure and primary infection at the surface of human palatine tonsil. We believe that the human organ culture system can be manipulated to provide unique insights into the process of HIV exposure in the oral cavity, and that this insight will be invaluable in guiding the design of novel strategies to protect mucosal surfaces from both cell-free HIV virions and cell-associated HIV.
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Materials and Methods |
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Palatine Tonsil Tissue Samples
Tissues were obtained from routine tonsillectomies performed at the Fairview University Medical Center (FUMC), Minneapolis, Minnesota with the assistance of the FUMC Tissue Procurement Facility. The experimental protocols had full Institutional Review Board approval, and individual patient consent for the use of tissue in research applications was obtained prior to initiation of the surgery. Pieces of palatine tonsil were routinely delivered within 13 hr of completion of surgery, and any tissue with recognizable macroscopic abnormality was automatically rejected. The tissue was randomly cut into small pieces (13-mm cubes) with either scissors and forceps or a pair of scalpel blades. The resultant tissue pieces were cultured on nitrocellulose membranes (0.4-µm pore size) placed on top of collagen supports (Gelfoam; Upjohn/Pharmacia, Kalamazoo, MI) or on Falcon transwell membranes (0.4-µm pore size; Becton Dickinson Labware, Franklin Lakes, NJ) at the gaseous/liquid medium (complete RPMI) interface in a standard CO2 incubator (Figure 1). Tissue pieces on transwell membranes supported the outgrowth of primary epithelial cells. On a historical note, our methods for the outgrowth of tonsillar epithelial cells and culture of small tonsil pieces are actually related to procedures that were first published many years ago (Shapiro and Volsky 1983
; Ferro et al. 1993
). Single-cell suspensions of tonsillar cells, comprised predominantly of B and T lymphocytes, were prepared by forcing small tissue pieces through a wire mesh with a syringe plunger as is routinely performed to disrupt mouse spleen. After extensive washing, the released cells were either cultured in complete RPMI or resuspended in fetal calf serum + 10% DMSO and frozen in liquid nitrogen. In some experiments, suspensions of freshly disrupted tonsil cells were purified away from erythroctyes by banding on Ficoll gradients (Histopaque 1077; Sigma, St Louis, MO).
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PCR Amplification of HIV Target Sequences
Randomly cut pieces of tonsil were positioned on nitrocellulose membranes on top of collagen supports, and HIV infections were initiated with cell-free virus stocks of the primary patient isolate, HIV96-480. The infected tissue pieces were incubated at the gaseous/liquid interface and, at various times after infection, tissue pieces were removed from the membranes and immediately disrupted in Trizol (Invitrogen; Carlsbad, CA). Total RNA was recovered in accordance with the manufacturer's recommended procedure. cDNA was synthesized using a random primer (Invitrogen), and PCR amplifications were performed under standard hot-start conditions with conserved primers from the HIV-1 gag coding region: forward primer: 5'-GTCAGCCAAAATTACCCTATAGTGCAGAAC; reverse primer: 5'-ACATAGTCTCTAAAGGGTTCCTTTGGTCCT with 2.5 mM MgCl2, and 40 two-step cycles (94C, 45 sec; 72C, 120 sec) followed by a final extension for 10 min at 72C. Amplification products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining and UV illumination.
Immunocytochemistry and Immunofluorescence
The following antibodies were used at a 1:100 dilution: HIV p24 (clone Kal-1; DAKO, Carpinteria, CA), S100 (rabbit polyclonal antibody, DAKO), Ki67 (Novocastra Laboratories; Newcastle, UK), and CD45RO (clone UCHL-1; Biogenex, San Ramon, CA). Negative controls for immunocytochemistry and immunofluorescence were appropriate for the primary antibody and confirmed specific detection of the desired antigen: HIV p24, matched, non-infected tissue; S100, omission of the primary antibody; and CD45RO, an isotype control antibody (mouse IgG; Vector Laboratories, Burlingame, CA). Secondary antibodies were conjugated to biotin for ABC amplification (Vector Laboratories) or cyanine 3 and cyanine 5 (Jackson ImmunoResearch Laboratories; Westgrove, PA) for fluorescent signals. Standard detection procedures were used; briefly, samples were prepared, processed through antigen retrieval and peroxidase blocking steps as needed, blocked with 1.5% serum, and incubated with the appropriate primary antibody overnight at 4C. Samples were then washed, re-blocked, and incubated with the appropriate secondary antibodies and detection systems for 1 hr at room temperature and washed and mounted in Permount and stored at room temperature or, for immunofluorescence, samples were mounted in Vectashield Hardset (Vector Laboratories) and stored at 4C until viewed.
Confocal Microscopy
Fluorescent images were collected with a Bio-Rad 1024 Laser Scanning Confocal microscope (Bio-Rad; Hercules, CA) equipped with a krypton/argon laser and processed with Confocal Assistant (written by Todd Clerke Brelje, University of Minnesota, Minneapolis, MN) and Adobe Photoshop (Adobe Systems Inc.; San Jose, CA). The time-lapse images were obtained by serial collection of a z-series repeated at 4-min increments.
Scanning Electron Microscopy (SEM)
Tissue was fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 10 hr, rinsed twice in the same buffer, and postfixed in 1% osmium tetroxide, again in the same buffer, for 45 min. The samples were then dehydrated through a graded series of alcohols, critical-point dried from liquid CO2, and coated with 23 nm platinum by indirect ion-beam sputtering before examination in a Hitachi S-4700 field-emission SEM (Hitachi High Technologies America; Pleasanton, CA).
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Results |
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Additional independent confirmation of HIV96-480 replication within cut tissue pieces was obtained from a combined immunocytochemistry/in situ hybridization procedure (Zhang et al. 1999). Cytoplasmic HIV RNA was observed in both activated T cells (Ki67+ cells) and resting T cells (Ki67 cells) in the tonsil organ culture system (Figure 3B). This finding exactly matched the results obtained from a rhesus macaque model for SIV sexual transmission, wherein SIV replication was detected in both activated and resting T cells in proximity to exposed mucosal surfaces and in the draining lymph nodes of vaginally infected macaques (Zhang et al. 1999
). The susceptibility of tonsillar lymphocytes to direct HIV infection was also demonstrated using tonsil-cell suspensions. Following exposure to cell-free HIV96-480, infected T cells were readily detected at day 7, in the absence of any exogenous cytokine stimulation of the tonsillar lymphocyte population (Figure 3C). The role of an intact epithelium in impeding the initiation of primary HIV infection was demonstrated by comparing adjacent pieces of tissue from the same tonsil. These tissue pieces were embedded in agarose to cover all cut tissue surfaces (Maher et al. 2004
), and then the external epithelial surfaces were damaged to varying degrees prior to exposure to HIV96-480 virions. In these experiments,
5- to 10-fold more infected cells were detected in tissue pieces either lacking epithelium or with damaged epithelium at the time of infection when compared with tonsil pieces that retained an undamaged epithelial surface during exposure to infectious HIV (Figures 3E3G).
To begin to reconstruct elements of biological complexity that are associated with natural routes of HIV-1 transmission, we have attempted to transfer HIV infection into tonsillar T cells using semen from HIV-seropositive individuals. Because of concerns regarding potential cytotoxic properties of seminal plasma (Fiore et al. 1997a), we have routinely separated seminal fluid into two fractions: seminal cells and cell-free seminal plasma. Coculture of viable seminal cells (a mixture of mature spermatozoa, immature round germ cells, and leukocytes) with a single-cell suspension of primary tonsillar cells resulted in transfer of HIV infection into the tonsillar T-cell population and accumulation of p24 antigen in the culture supernatant. Soluble p24 levels increased 100-fold between day 5 (17.5 pg/ml) and day 17 (1.75 ng/ml) of culture for the exposed tonsillar lymphocyte populations. There was no detectable accumulation of p24 when matched populations of tonsillar cells were exposed to cell-free seminal plasma from the same semen donor. In subsequent experiments, we determined that 80% to 90% of tonsillar lymphocytes were killed by overnight incubation in seminal plasma (10% v/v seminal plasma in complete RPMI; 22 hr at 37C in a standard CO2 incubator). Thus, oral exposure to HIV infectivity in semen may involve a balance between direct cytotoxic effects of seminal plasma and cell-to-cell interactions that can successfully transfer HIV infectivity into tonsillar T cells.
As another indicator of tonsillar lymphocyte susceptibility to HIV infection and the ability of infected tonsillar lymphocytes to release infectious progeny HIV virions, we used the day 17 (1.75 ng/ml p24) filtered tissue culture supernatant described above as a virus inoculum to transfer HIV into new tonsillar lymphocyte suspensions, derived from two completely independent adult donors. In both cases it was possible to observe the time-dependent appearance of HIV infection in the exposed tonsil-cell populations (p24 immunocytochemical staining of infected T cells, data not shown). This demonstration of the production of infectious HIV within tonsillar lymphocyte populations provides a useful experimental correlate for the observation of HIV shedding in the oral cavity of some HIV-seropositive individuals (Zuckerman et al. 2003) because infected tonsillar T cells could be regarded as a potential source of HIV to contribute to oral shedding.
Collectively, the preceding results highlight the importance of a fully intact tonsillar epithelium to provide a physical barrier between HIV virions and the otherwise readily susceptible tonsillar lymphocytes. However, as part of an ongoing survey of the morphological characteristics of tonsillar epithelium, we have examined a representative series of tonsils that were immediately fixed on receipt into the laboratory. Somewhat surprisingly, localized irregularities, including lymphocyte invasion of the epithelium, were commonly observed in the stratified squamous epithelium covering the external surface of the palatine tonsil in 10 of 11 randomly selected tonsils (Figure 4A). Immunocytochemical staining with an antibody directed against CD45RO (clone UCHL-1) (Janossy et al. 1989) confirmed that activated T cells could readily be detected within and immediately below the stratified squamous epithelium (Figure 4B). Furthermore, in examining tonsil pieces by SEM, it was possible to identify occasional distinctive cells located at the tonsil surface and to recognize an abrupt transition in the topographical structure of the surface (Figures 4C and 4D). Considered together, these structural aberrations may have profound implications for HIV transmission by allowing potentially susceptible lymphocyes to be situated in proximity to infectious inocula that had been deposited onto the external surface of the tonsil.
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In a final series of experiments, we have used confocal microscopy to begin to describe the sequence of events that unfolds after HIV exposure at mucosal surfaces. HIV-infected cells could bind stably to the epithelial surface of live tonsil pieces (Figure 6A), and in this image the infected lymphocytes are positioned at the entrance to a crypt, in proximity to S100-positive dendritic cells situated sporadically throughout the palatine tonsil (Noble et al. 1996; Papadas et al. 2001
; Maeda et al. 2002
). Although dendritic cells have been recognized as participating in one uptake mechanism for cell-free HIV virions through virion binding to DC-SIGN (Geijtenbeek et al. 2000
; Pohlmann et al. 2001
), the role of dendritic cells in the uptake of HIV-infected foreign cells is far less defined. The dynamic complexity of cellcell interactions at epithelial surfaces was best revealed by time-lapse confocal microscopy. We observed some lymphocytes migrating across the epithelial surface and other lymphocytes stably bound to the epithelial surface within 60 min of exposure (Figure 6B). We are currently attempting to identify the ligandreceptor interactions that may facilitate lymphocyte binding to epithelial surfaces, but we have observed that fluorescently labeled latex spheres (4-µm diameter) can also bind to epithelial surfaces or become trapped on epithelial surfaces (data not shown). However, it is extremely unlikely that the cell distortions visible as cells migrated across the tonsil surface could reflect nonspecific interactions (Figure 6B). In the context of HIV transmission it must be recognized that both specific and nonspecific mechanisms may contribute to lymphocyte binding and retention at epithelial surfaces and that such binding may be a prelude to transfer of HIV infectivity across an otherwise intact mucosal barrier.
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Discussion |
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In total, the findings reported here provide strong support for the identification of tonsillar tissue as a portal of entry for HIV infectivity and site for primary HIV infection, following oral exposure. Several other viruses, including polio virus, JC virus, cytomegalovirus, EpsteinBarr virus (EBV), Kaposi's sarcoma herpes virus, and human herpes virus 6, are believed to use tonsil as the site of primary infection (Goudsmit et al. 1982; Monaco et al. 1996
; Mbopi-Keou et al. 2002
). These viruses probably employ different mechanisms to traverse the tonsillar epithelium, but for EBV there is some evidence that tonsillar epithelial cells may be susceptible to productive virus infection (Borza and Hutt-Fletcher 2002
). In the same general context, it is interesting to note that tonsillar inflammation has been implicated as one possible mechanism whereby consumption of meat products from cattle affected by bovine spongiform encephalopathy could lead to the onset of variant CreutzfeldJakob Disease in young adults (Nailon and Ironside 2000
; Hilton et al. 2002
).
We believe that multiple mechanisms may account for HIV transmission across the tonsillar epithelium, and that there will be a recognizable connection between the specific properties of the HIV inoculum (cell-free virions or cell-associated infectivity) (Crittenden et al. 1992; Quayle et al. 1997
; Fiscus et al. 1998
), the macro- and microarchitecture of the target tissue at the exposed sites, and the mechanism of infection transfer. If the epithelium has been transiently damaged either by recent trauma or by previous or ongoing microbial infection, then HIV infectivity may gain direct access to tonsillar leukocytes in a process that is largely analogous to our experimental infection of cut tonsillar tissue pieces in organ culture (Figure 2 and Figures 3A, 3B, and 3E). With cell-associated infectivity, HIV-infected cells in the inoculum could fuse to surface epithelial cells (Figure 5) and then, at least for reticulated epithelium, release of virions from the basolateral epithelial cell surface would position HIV infectivity in immediate proximity to CD4+ leukocytes. In addition to fusion at the epithelial surface, infected donor cells may even invade the epithelium. Although any such invading donor cells should rapidly be removed by phagocytosis, the process of local recruitment of activated recipient cells in response to recognition of foreign antigens may serve to facilitate the initiation of primary HIV infection. Any temporary disruption or relaxation of tight junctions formed between epithelial cells could allow paracellular transport of virions or infected cells across the epithelial surface. The process of transcytosis has been carefully described for transfer of HIV virions across monolayers of cultured epithelial cell lines in vitro (Bomsel 1997
; Belec et al. 2001
), but it is far from clear if transcytosis is involved in HIV uptake in natural infections. Any one of these distinct uptake mechanisms may be sufficient to establish primary HIV infection at an exposed mucosal surface. However, the combination of different uptake mechanisms with the morphological irregularities that we have observed in epithelial surfaces can be projected to lead to increased susceptibility for primary HIV infection.
On one level of thinking, much of the HIV transmission in the ongoing global HIV pandemic is readily understandable because virus is spread from person to person either by intimate sexual contact or by exposure to infected blood. In the case of mucosal exposure to HIV infectivity, there appears to be considerable protective benefit from an intact epithelial surface. Conversely, physical trauma or preexisting microbial infections can cause extensive change in the characteristics of mucosal surfaces and lead to elevated susceptibility to primary HIV infection. There is a clear association between STDs in the female reproductive tract and acquisition of primary HIV infection and similar connections between preexisting infections and HIV infection can be projected to occur in the oral cavity (Fiore et al. 1997b; Hillier 1998
; Sturm-Ramirez et al. 2000
). In this respect, it must be noted that the tonsil tissue used in these experiments cannot necessarily be considered as "normal." Most of the tissues showed mild to moderate hyperplasia and had been removed from individuals with chronic tonsillitis or sleep apnea. In both clinical conditions, inflammation may have contributed significantly to tonsillar enlargement. The combination of alterations in the mucosal surface and activation and redistribution of tonsillar lymphocytes may dramatically enhance susceptibility to primary HIV infection, following oral exposure. Looking into the future, we anticipate that human mucosal organ culture systems will support a unique role in the evaluation of new protective strategies designed to curtail the spread of HIV and that the binding events and surface aberrations we have described will need to be addressed with the goal of achieving comprehensive protection against HIV.
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Acknowledgments |
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We would like to thank our Microbiology colleagues, Dr. Ashley Haase, Dr. Hae-Sun Park, Dr. Kathryn Staskus, Dr. Pamela Skinner, and Stephen Wietgrefe, and Dr. Stefan Pambuccian in the Department of Surgical Pathology, University of Minnesota Medical School, for invaluable advice and insightful critiques. This work would not have been possible without the skillful assistance of Ms. Diane Rauch and Ms. Sarah Bowell in the Tissue Procurement Facility, Fairview University Medical Center (FUMC), Minneapolis, Minnesota, and Matthew Larson in the Department of Medicine (FUMC) who were collectively responsible for the primary interactions with the patients. We greatly appreciate the willingness of many anonymous patient donors who have provided samples for research purposes. We also acknowledge general technical assistance provided by Ms. Julie Horbul, SEM instruction and assistance provided by Chris Frethem, and essential contributions provided by the staff and resources within the Bioimaging and Processing Laboratory and the Supercomputing Institute at the University of Minnesota.
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Footnotes |
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