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Journal of Virology, October 1998, p. 7822-7829, Vol. 72, No. 10
Laboratoire de Biologie et
Thérapeutique des Pathologies Immunitaires,
Received 3 April 1998/Accepted 26 June 1998
We have developed a murine model to study the involvement of
dendritic cells (DC) in human immunodeficiency virus (HIV) routing from
an inoculation site to the lymph nodes (LN). Murine bone marrow-derived
DC migrate to the draining LN within 24 h after subcutaneous
injection. After incubation of these cells with heat-inactivated (Hi) HIV type 1 (HIV-1), HIV RNA sequences were detected in the draining LN only. Upon injection of DC pulsed with infectious HIV, the
virus recovered in the draining LN was still able to productively
infect human T cells. After a vaginal challenge with Hi HIV-1, the
virus could be detected in the iliac and sacral draining LN at 24 h after injection. After an intravenous challenge, the virus could be
detected in peripheral LN as soon as 30 min after injection. The
specific depletion of a myeloid-related LN DC population, previously
shown to take up blood macromolecules and to translocate them into the
LN, prevented HIV transport to LN. Together, our data demonstrate the
critical role of DC for HIV routing to LN after either a vaginal
or an intravenous challenge, which does not require
their infection. Therefore, despite the fact that the mouse is not
infectable by HIV, this small animal model might be useful to test
preventive strategies against HIV.
Understanding the early events that
lead to establishment of a chronic human immunodeficiency virus (HIV)
infection is essential for deciphering HIV pathophysiology and for
designing therapeutic and vaccination strategies against this virus.
HIV infection occurs through two main routes: sexual transmission, and
direct intravenous inoculation. The establishment of a chronic
infection requires that the contaminating virus productively infects
one of its target cells, which are CD4-expressing T lymphocytes
(22, 55), monocytes (13, 44), and dendritic cells
(DC) (8, 23, 25, 53). Recently, a series of arguments has
pointed to a scenario for the establishment of primary HIV infection
through sexual transmission which involves CD4+ T
lymphocytes and DC (23, 36, 43, 49, 50). It is assumed that
DC at the inoculation site are first infected by HIV and then
migrate to the draining lymph nodes (LN), where they transmit the virus
to T cells in paracortical zones. Intense replication ensues, followed
by dissemination of the virus to the whole lymphoid system. This
scenario is inferred from (i) the fact that mucosae contain Langerhans
cells (LC), immature DC known to migrate from their resident area to
the draining LN upon exposure to antigens or to inflammatory signals
(17-19, 24, 31, 32, 34, 46); (ii) observations of the
nature and location of simian immunodeficiency virus (SIV)-infected
cells at different time points after an experimental SIV vaginal
inoculation in macaques (50); and (iii) in vitro observations in which conjugates of DC and T cells represent the optimal milieu for a productive HIV infection, because DC transmit HIV
to T cells together with a vigorous activation signal that permits
efficient infection and replication (9, 42). Although this
scenario appears likely, the role of DC has not been definitively demonstrated. As pointed out previously (50), the first
infected cells are seen in the lamina propria after an SIV vaginal
inoculation, and it has not yet been explained how the virus crosses
the multilayered epithelium that usually acts as a physical barrier to
infectious agents. It is also not known how HIV reaches the LN in the
case of an intravenous (i.v.) inoculation.
We aimed to analyze in more detail the role of DC in the early events
of HIV infection. Because studies using primates and infectious virus
are difficult to carry out, we investigated the possibility of using a
mouse model. Indeed, although murine cells are not infectable by HIV,
mice may serve as a good model to study HIV routing by DC. First,
murine DC are well characterized and can be easily traced with specific
antibodies (51). Second, after staining and reinjection,
migration of these cells can be easily monitored (3, 16, 21, 26,
37), and there are solid experimental data on the circulation
pathways of murine DC (2). Finally, it has previously been
reported that murine DC, although not infectable by HIV, can transmit
HIV to human T cells as efficiently as human DC (9). This
indicates that DC can transmit to T lymphocytes HIV particles that they
have not themselves produced but rather that they have adsorbed or internalized.
We show here that infectious HIV can indeed be transported by murine DC
to the draining LN upon inoculation at the periphery and that DC are
also essential for carrying HIV to LN upon i.v. administration. These
results indicate that DC play a major role in the establishment of HIV
infection which does not necessarily require their productive
infection.
Animals.
DBA/2 and (C57BL6 × CBA/J)F1 mice were purchased from IFFA-Credo (L'Arbresle,
France). Human CD4 (hCD4)-transgenic mice derived from
(C57BL6 × CBA/J)F1 mice were obtained from P. Lorès (29). Mice were 7 to 14 weeks of age at the time
of the experiments. Thymidine kinase (TK)-chimeric mice were derived
from LTR-TK-transgenic mice expressing the herpes simplex virus type 1 TK (HSV-1 TK) in DC as previously described (47, 48).
Preparation of DC.
DC were obtained from bone marrow (BM) of
DBA/2 or hCD4-transgenic mice as described previously (20),
with slight modifications. Briefly, BM cells were collected from femurs
and tibias, erythrocytes were lysed with ammonium chloride, and
immunomagnetic depletion of lymphocytes and Ia-positive cells was
performed with a mixture of hybridoma supernatants of anti-CD4
(GK-1.5; ATCC TIB207), anti-CD8 (H35 JP), anti-major
histocompatibility complex (MHC) class II (M5/114; ATCC TIB120), and
anti-B220 (RA3-6B2) monoclonal antibodies (MAb) and sheep anti-rat
immunoglobulin G (IgG)-coated Dynabeads (M-450; Dynal, Oslo, Norway).
After the depletion steps, cells were cultured at 7 × 105 cells/ml in RPMI 1640 supplemented with 5%
decomplemented fetal calf serum (FCS), 20 µg of gentamicin per ml, 50 µM 2-
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Dendritic Cells Route Human Immunodeficiency Virus to Lymph Nodes
after Vaginal or Intravenous Administration to Mice
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-mercaptoethanol, and 2,000 U of recombinant
granulocyte-macrophage colony-stimulating factor (GM-CSF) (Genzyme,
Cambridge, Mass.) per ml in 24-well plates at 37°C. On days 2 and 4, 90% of the medium was replaced by GM-CSF-containing medium. On day 6, the medium was harvested, and loosely adherent aggregates of growing DC
were dislodged by vigorous pipetting and then counted (47).
DC were subcultured at 2 × 105 to 3 × 105 cells/ml in fresh GM-CSF-containing medium in six-well
plates overnight and then harvested and counted.
Cell line. The CD4+ human lymphoid cell line HUT-78 was purchased from the American Type Culture Collection (Rockville, Md.) and maintained in RPMI 1640 supplemented with 10% decomplemented FCS (Dutscher, Brumath, France).
HIV strains. The lymphotropic HIV type 1 (HIV-1) strain HIV-1LAI (52) and the macrophage-tropic HIV-1Ba-L were obtained from Diagnostics Pasteur (Marne-la-Coquette, France) and from B. Asjö, respectively. The HIV-1LAI titer, determined by infection of activated peripheral blood mononuclear cells (PBMC) with viral supernatant, was 104 50% tissue culture infective doses (TCID50)/ml. The concentration of p24 antigen in the HIV-1LAI viral supernatant was 5 µg/ml.
Live virus supernatant was used either for subcutaneous (s.c.) injection (25 µl = 250 TCID50) or for incubation experiments (100 to 300 µl [3 × 106 to 10 × 106 cells/ml] = 1,000 to 3,000 TCID50). Other experiments were performed after heat inactivation (Hi) of the virus at 56°C for 1 h. In this case, because the virus inoculum cannot be evaluated as TCID50 per milliliter, viral amounts are given as the volume of either pure or diluted supernatant. HIV-1Ba-L, with a titer of 105 TCID50/ml determined by infection of activated PBMC with viral supernatant, was used after similar Hi.In vivo tracing of DC. (i) s.c. injection of BM-derived DC. The migration pattern of BM-derived DC was analyzed by injecting cells stained with PKH2 (Zynaxis, Malvern, Pa.), a nontoxic fluorochrome previously used in migration studies of murine antigen-presenting cells (37). BM-derived DC suspended at a final concentration of 5 × 106 to 10 × 106 cells/ml in a diluent (Zynaxis) were incubated for 5 min at room temperature with 2 µM (final concentration) PKH2. Two volumes of RPMI 1640 with 10% fetal calf serum was added to stop the reaction, and then the cells were washed three times.
PKH2-stained and control cells were injected s.c. at 5 × 105 cells/25 µl into the footpads of anesthetized (2.5% tribromoethanol) DBA/2 mice. At 6 and 24 h after injection, ipsilateral and contralateral popliteal and inguinal LN were harvested, frozen in OCT medium (Labonord, Villeneuve d'Ascq, France), sectioned (5 µm) on a cryostat, and viewed under a fluorescence microscope.(ii) FITC application on the vaginal mucosa. The migration of resident LC present in the vaginal mucosa was investigated by using a previously published technique utilized for studying the migration of epidermal LC (31, 46). Twenty-five to 30 µl of 0.8% FITC (Isomer 1; Sigma Chemical Co.) dissolved in phosphate-buffered saline was applied atraumatically by inserting a 22-gauge 1 1/2 animal feeding cannula (Polylabo, Strasbourg, France) onto the vaginal vaults of anesthetized mice. The animals were maintained immobilized for 30 to 45 min. Vaginal mucosae and sacral, iliac, lumbar, and inguinal LN were harvested 12 and 24 h later for flow cytometry analysis.
Coculture experiments. DC (3 × 106 to 10 × 106) were incubated with 100 µl of either infectious or Hi HIV-1LAI supernatant in a final volume of 1 ml of complete medium for 2 h at 37°C and washed twice in RPMI, and then 5 × 104 DC were cocultured with 5 × 105 HUT-78 cells in RPMI 1640-10% FCS for 10 days. Controls were unpulsed DC.
Similarly, HIV-pulsed DC were cocultured with 5 × 105 T cells isolated from PBMC by rosetting in the presence of 100 ng of staphylococcal enterotoxin A (SEA) per ml in RPMI 1640 supplemented with 10% AB+ normal human serum, glutamine (2 mM), and 1% antibiotics (GIBCO-BRL, Paisley, Scotland) for 10 days. Controls were pulsed T cells or unpulsed DC cocultured with unpulsed T cells. Cells obtained after mechanical dilaceration of LN were cocultured with 5 × 104 and 1 × 105 HUT-78 cells for popliteal and inguinal nodes, respectively, in RPMI 1640-10% FCS with glutamine and antibiotics. Cells were expanded for 10 days and then maintained at 105 cells by biweekly medium changes up to 80 days. The kinetics of viral p24 production in the different culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA) (Diagnostics Pasteur).In vivo HIV-1 experiments. (i) s.c. injection. In a first set of experiments, infectious or Hi HIVLAI supernatant (25 µl/mouse) was injected s.c. into the footpads of anesthetized DBA/2 mice. At 24 h after injection, ipsilateral, contralateral popliteal, and inguinal LN were removed and frozen for RNA extraction.
In another set of experiments, 3 × 106 to 10 × 106 BM-derived DC were first incubated with 300 µl of either infectious or Hi HIVLAI or HIVBa-L supernatant in a final volume of 1 ml of complete medium for 2 h at 37°C. After two washes, 5 × 105 cells/25 µl were injected s.c. into the footpads of anesthetized mice. At 24 h after injection, ipsilateral, contralateral popliteal, and inguinal LN were removed and frozen for RNA extraction. The amount of virus bound to BM-derived DC before their injection was determined by p24 ELISA.(ii) Application on the vaginal mucosa. Twenty-five microliters of Hi HIV-1LAI supernatant was administered in the vaginal vaults of anesthetized mice as described above for FITC. Twenty-four hours later, vaginal mucosae and sacral, iliac, lumbar, and inguinal LN were harvested and frozen for RNA extraction.
(iii) i.v. administration. One hundred fifty microliters of Hi HIV-1LAI supernatant was administered i.v. into the retroorbital sinuses of anesthetized mice. Thirty minutes later, peripheral LN (axial, brachial, and inguinal) were harvested separately and then mixed into two pools corresponding to the left and right LN of mice. The kidney was also harvested as a control. All of the samples were frozen for RNA extraction.
RT-PCR. Control and HIV-pulsed cells were lysed in RNA extraction solution (RNA-BTM; Bioprobe, Montreuil, France). Frozen LN and kidney were teased with a scalpel in the same solution.
For the reverse transcription-PCR (RT-PCR), 1 µg of total cellular RNA or half total cellular RNA of LN was reverse transcribed as follows. In a 20-µl reaction mixture, 1 mM each deoxynucleoside triphosphate (Pharmacia LKB Biotechnology), 0.04 U of random primer P(dN)6 (Pharmacia LKB Biotechnology), 40 U of RNase inhibitor (Pharmacia LKB Biotechnology), 10 mM dithiothreitol, and 200 U of Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Gaithersburg, Md.) were used. RT reactions were performed at 42°C for 1 h. A 5-µl aliquot of the RT mixture was used directly for each amplification reaction. PCR was performed as follows. In a 50-µl reaction mixture, 0.5 µM primers SK38 and SK39 for HIV-1 gag sequences (Perkin-Elmer Cetus, Emeryville, Calif.) or 1 µM hCD4 primers (Eurogentec, Angers, France), 200 µM each deoxynucleoside triphosphate (Pharmacia LKB Biotechnology), 1.5 mM MgCl2, and 1 U of Taq DNA polymerase (Goldstar DNA polymerase; Eurogentec, Seraing, Belgium) with 1× buffer were used. The hCD4 primers were as follows: sense primer, 5'-ATGAACCGGGGAGTCCCTTTT-3'; antisense primer, 5'-ACTATCCTGGAGCTCCAGCT-3'. Reaction mixtures were incubated in a Crocodile II DNA thermal cycler (Appligene, Illkirch, France). For the HIV-1 gag primers, after 5 min at 94°C, 35 cycles of denaturation for 60 s at 94°C, annealing for 60 s at 58°C, and extension for 60 s at 72°C were performed, followed by an extension of 10 min at 72°C at the completion of the run. For the hCD4 primers, after 5 min at 94°C, 35 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 64°C, and extension for 60 s at 72°C were performed, followed by an extension of 10 min at 70°C. A second amplification was performed under the same conditions with 5 µl of 1:200 diluted PCR products as the template. The products of PCR were analyzed by Southern blot hybridization with the HIV-1 SK19 probe (Perkin-Elmer Cetus). For quantitative RT-PCR, the test Amplicor HIV-1 Monitor (Roche Diagnostic Systems, Neuilly-sur-Seine, France) was used with some modifications: this quantification was performed not with plasma but with 2 µg of RNA extracted from LN with RNA extraction solution (RNA-BTM; Bioprobe) and treated with RNase-free DNase (Boehringer Mannheim, Meylan, France).| |
RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Murine BM-derived DC efficiently transmit HIV-1 infection to human T cells. Murine DC can routinely be generated from BM cells cultured in the presence of GM-CSF for 7 days (20). Under such culture conditions, 40 to 70% of the cells were DC as characterized by their morphology and phenotype, i.e., expression of the CD11c marker and of high levels of MHC class II molecules (20, 30, 47).
We verified that these BM-derived DC could efficiently transmit productive HIV infection to human T cells as previously reported for murine splenic DC (9). After incubation with either Hi or infectious HIV-1LAI, DC were cocultured with HIV-susceptible human HUT-78 cells. The cells became productively infected (Fig. 1A), demonstrating that the virus bound to murine BM-derived DC remained infectious. Similarly, DC could transmit an HIV infection to human resting T lymphocytes when these cells were cocultured in the presence of a superantigen (Fig. 1B). In contrast, resting T lymphocytes incubated with HIV-1 and a superantigen in the absence of DC did not replicate HIV-1.
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Murine BM-derived DC migrate to the draining LN after s.c. injection. Peripheral DC are known to migrate to the draining LN upon antigen stimulation. BM-derived DC can similarly migrate to the draining LN after s.c. injection. Fluorescent BM-derived DC labeled with the PKH2 tracer were already found in the draining ipsilateral popliteal LN 6 h after injection, and their number increased at 24 h (Fig. 2A). No fluorescent cells could be detected in the ipsilateral inguinal or the contralateral popliteal and inguinal LN. Thus, BM-derived DC generated in vitro retain their ability to migrate from an s.c. injection site to the draining LN.
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Murine BM-derived DC transport HIV to draining LN after s.c. injection. We then repeated these experiments with DC incubated with the lymphotropic Hi HIV-1LAI. At 24 h after s.c. injection of Hi HIV-1LAI-pulsed DC into the footpad, HIV gag RNA sequence was detected in the ipsilateral popliteal nodes of three of five mice, but it was not found in the ipsilateral inguinal or in the contralateral popliteal and inguinal LN (Table 1). In experiments performed with the macrophage-tropic Hi HIV-1Ba-L, HIV gag RNA sequence was also found only in the ipsilateral popliteal LN (data not shown).
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HIV is still infectious after transport to LN. Similar experiments with infectious virus allowed us to evaluate whether it remained infectious upon arrival in the LN. After injection of infectious HIV-1LAI, draining popliteal LN cells were harvested and cocultured with HIV-susceptible human HUT-78 cells. A productive HIV infection of these cells was detected in one of four mice injected with HIV-1LAI-pulsed DC and in one of four mice injected with 25 µl of highly concentrated HIV-1LAI supernatant (Fig. 3). Thus, HIV can remain infectious 24 h after transport from its injection site to the draining LN in mice.
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HIV is transported to draining LN after vaginal application. Previous reports showed that LC of the vaginal mucosa can pick up antigens and migrate to the draining LN (39, 41). In agreement with these studies, we observed FITC-positive cells only in draining sacral and/or iliac LN 24 h after FITC was applied on the vaginal mucosa. No positive cells were observed in inguinal LN (Fig. 4A). The stained cells were detected within an LN DC subset (l-DC) (Fig. 5) previously shown to derive from LC (46).
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DC participate in the transport of HIV to LN after i.v. inoculation. We recently showed that a myeloid-related DC subset of peripheral LN (s-DC) (Fig. 5) has the ability to take up blood macromolecules and transport them to LN (46). We thus aimed to analyze if they could also route viral particles from the blood compartment to LN.
We injected Hi HIV-1LAI supernatant i.v. into normal mice, and 30 min later, we studied HIV gag RNA in pools of peripheral LN, as well as in the kidney, by RT-PCR. Because in this case the viral inoculum would be immediately diluted within the blood compartment, we injected a larger volume of viral supernatant (150 µl) than in the experiments described above. The HIV gag RNA signal was strong in six of eight and weak in two of eight pools of peripheral LN (Table 2). In all cases, the kidney, which was chosen as a positive control tissue because of its high blood content, was also found to be positive for HIV gag RNA. Although this is unlikely, the positive LN signals could have been due to blood contamination. To rule out this hypothesis, we repeated these experiments using mice specifically depleted of s-DC. We used our model of transgenic mice preferentially expressing an HSV-1 TK gene in DC (1, 27, 28, 46-48). Ganciclovir (GCV), which is specifically metabolized into a toxic analog in HSV-1 TK-expressing cells, kills dividing HSV-1 TK-expressing DC. As shown in Fig. 5, a 7-day GCV treatment dramatically affected s-DC, which was reduced approximately 10-fold (from 0.2 to 0.4% of total LN cells in control mice to 0.03 to 0.05% in DC-depleted mice) (46). By contrast, this treatment did not affect l-DC, in accordance with their turnover, and the overall LN architecture was not affected (27a). To ascertain the role of s-DC in Hi HIVLAI routing to LN, we performed i.v. injections of Hi HIVLAI in s-DC-depleted mice. As shown in Table 2, no HIV gag RNA was detected in five of eight LN cell pools, while a weak signal was detected in two of eight pools and a strong signal was detected in one of eight pools. These results were confirmed by using a quantitative RT-PCR kit used for clinical HIV-1 detection, with RT-PCR performed on the total peripheral LN cells from control and DC-depleted mice (Table 2). Similar results were obtained in a second, independent experiment.
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Intravaginal inoculation of SIV in macaques (50) revealed a role for DC in the initiation of a primary infection. These experiments clearly showed that after an initial viral challenge, the virus is rapidly (in less than 2 days) detectable in the draining LN. On day 2, the virus is already disseminated, as illustrated by its detection in the spleen and nondraining LN (50). Thus, the kinetics and the localization of these events correlate well with the known DC migration pattern and physiological interaction with T cells. By inference, it was therefore assumed that DC at the inoculation site became HIV infected, migrated to the LN, and transmitted HIV infection to local T cells that thereafter became the seeding point for viral dissemination. However, these experiments also raised questions regarding the precise role of DC. First, productively SIV-infected cells were detected only in the lamina propria of the cervicovaginal mucosa and not in the epithelium. Thus, how did the virus penetrate into the lamina propria, and are other cells involved in this initial step of SIV infection? Second, these infected cells have not be formally identified as DC. Third, SIV could be detected in the vagina-draining LN in only one of the four animals studied. Therefore, it remains to be formally demonstrated if and which DC are the initially infected cells after sexual exposure to HIV and are responsible for its systemic dissemination. However, these experiments are difficult to carry out and expensive with large animals and infectious virus. This led us to investigate the possibility of using mice to analyze some of these events in more detail.
Although murine cells cannot be infected by HIV, the mouse model seemed appropriate to address some of these questions, in part because the migration patterns of murine BM-derived DC and chimpanzee DC are similar (5). Indeed, our results indicate that even in the absence of a productive infection, murine DC are able to transport HIV, regardless of the site of administration, from the periphery to the draining LN.
Several points should be emphasized. First, the role of DC in HIV routing after s.c. injection was demonstrated with DC purified by cell sorting. Second, the fact that the HIV-1 RNA was not detected in 100% of injected mice is most probably due to the sensitivity of the detection technique, which might be improved in the future. Furthermore, it should be pointed out that we never detected any viral signal in a single nondraining LN throughout the 10 independent experiments performed. This is also true for the experiments carried out with infectious HIV-1LAI, which could be found in the draining LN of two of eight animals tested. The detection, even on a single occasion, of live infectious HIV-1 in the LN demonstrates that it can be transported from the periphery to the LN while retaining its infectivity. Third, HIV transport by DC was shown for both lymphotropic and macrophage-tropic strains; the latter are presumed to be the predominant strains mediating primary infection in humans (4, 14). Fourth, our data indicate that DC could play a major role in HIV-1 dissemination without being productively infected. This is in agreement with previous in vitro observations that also suggested a role for DC in HIV transmission to T cells in the absence of their infection (9, 11). Finally, our results provide a possible explanation for the absence of infected DC in the superficial vaginal mucosae of the SIV-challenged macaques. We suggest that in primates susceptible to HIV or SIV infection, these viruses can be taken up by resident mucosal LC that would carry them to LN without being infected.
It is noteworthy that murine DC can bind and transport HIV-1 without abrogating virus infectivity. Interestingly, in vitro experiments showed that HIV incubated with DC could escape trypsin treatment and remain infectious for T cells (6, 10). It is thus possible that DC, either human or murine, bind and retain the virus on the cell membrane, where it could be sequestered within compartments formed by cell surface dendritic processes (6). Alternatively, the virus might be internalized into cytoplasmic vesicles.
It is noteworthy that the binding of HIV on murine DC did not require the hCD4 receptor. In addition, its presence did not significantly influence HIV transport by DC, as shown by results obtained with transgenic hCD4+ murine DC. Therefore, the HIV binding on DC must involve other mechanisms, possibly related to the glycosylation of the HIV envelope proteins and/or the existence of mannose receptors on DC (45). It has recently been shown that HIV-1 is able to bind to LC by a CD4-independent mechanism (6).
While HIV-1 transport from the mucosae to the LN involves the LC or DC, we show for the first time that HIV-1 transport from blood to LN appears to involve another recently characterized DC subset. These cells have an immature phenotype and appear to be myeloid related (46). Their unique capacity to take up blood molecules and to carry them to LN suggests that they play a major role in the immune response to blood antigens and microorganisms. In this case, it appears that this property is subverted by HIV to facilitate virus dissemination and the establishment of a chronic infection.
Together, our data strongly suggest that noninfected DC mediate the initial steps of HIV-1 infection after exposure through sexual transmission or direct i.v. inoculation. Furthermore, depending on the site of inoculation, different DC subsets are involved in HIV-1 uptake: local LC-derived DC (l-DC) upon sexual transmission or blood DC (s-DC) after i.v. injection. In both situations, the virus would be carried to LN, where extensive viral replication would occur upon its transmission to CD4+ T cells, in accordance with several reports (12, 15, 38). The resulting viral production would then lead to HIV dissemination throughout the lymphatic system.
We have developed a murine model to study the involvement of DC in HIV-1 routing. This model uses a small, widely available, and inexpensive animal. Moreover, the analysis of HIV-1 transport to LN does not require infectious HIV-1, thus dramatically simplifying the experimental conditions. In addition, it should now be possible to analyze HIV transmission from DC to LN T cells in a murine model. Indeed, T lymphocytes from double-transgenic mice expressing the hCD4 receptor and CCR5 coreceptor are susceptible to HIV infection (7). Therefore, murine models might be useful to test different factors influencing HIV-1 transmission (such as hormonal impregnation) (33, 40), to screen molecules that upon mucosal application could prevent HIV-1 transmission, and to test vaccines for their ability to trigger a mucosal immunity to attack HIV-1 at this early step of infection.
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ACKNOWLEDGMENTS |
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We thank Jean-Claude Gluckman for critical reading of the manuscript and Roger Lacave and Michelle Rosenzwajg for their help in some of the experiments.
This work was supported by the Université Pierre et Marie Curie,
the Assistance Publique
Hôpitaux de Paris, and the Centre National de la Recherche Scientifique. C.M. was supported by ARDIVI and
ARMO, and N.G. was supported by Sidaction.
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FOOTNOTES |
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* Corresponding author. Mailing address: CERVI, Groupe Hospitalier Pitié-Salpêtrière, 83 boulevard de l'Hôpital, 75651 Paris Cedex 13, France. Phone: 33-1-42-17-74-61. Fax: 33-1-42-17-74-62. E-mail: david.klatzmann{at}psl.ap-hop-paris.fr.
Present address: Committee on Immunology, The Ben May Institute,
University of Chicago, Chicago, Ill.
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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