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(Journal of Leukocyte Biology. 2001;70:313-321.)
© 2001 by Society for Leukocyte Biology

Functional HIV CXCR4 coreceptor on human epithelial Langerhans cells and infection by HIV strain X4

I. Tchou*, L. Misery*, O. Sabido{dagger}, C. Dezutter-Dambuyant{ddagger}, T. Bourlet*, P. Moja*, H. Hamzeh*, J. Peguet-Navarro{ddagger}, D. Schmitt{ddagger} and C. Genin*

* Groupe Immunité des Muqueuses et Agents Pathogènes and
{dagger} Centre de Cytométrie en Flux, University of Saint-Etienne; and
{ddagger} INSERM U346, Lyon, France

Correspondence: Prof. C. Genin, G.I.M.A.P., Faculté de Médecine, 15 rue Ambroise Paré, 42023 Saint-Etienne cédex 2, France. E-mail: geninc{at}univ-st-etienne.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HIV can cross the intact epithelium of genital mucosae via Langerhans cells. Fresh Langerhans cells are known to express CD4 and CCR5. The presence of CXCR4 on the surface of cultured but not freshly isolated Langerhans cells has been described. In the present study, we demonstrate that CXCR4 was expressed by fresh Langerhans cells isolated and purified from epidermis. However, the percentage of Langerhans cells expressing CXCR4 or CCR5 increased during maturation of the cells in culture, especially in the presence of exogenous granulocyte-macrophage colony-stimulating factor. To determine whether CXCR4 was functional, freshly isolated Langerhans cells were infected with HIV LAI, a T-cell-tropic strain, and p24 protein production was measured in culture supernatants. p24 production was observed when infected Langerhans cells were cocultured with SupT1 cells. However, the presence of HIV provirus DNA was evidenced within the infected Langerhans cells by nested PCR. Ultrastructural studies confirmed the formation of syncytia when Langerhans cells were cocultured with SupT1 cells. Preincubation of Langerhans cells with azidothymidine or SDF-1-{alpha}, a natural ligand for CXCR4, prevented infection. These data demonstrated that CXCR4 is present on the surface of Langerhans cells freshly isolated from human skin epidermis and that this expression is functional.

Key Words: HIV • Langerhans cell • CXCR4 • CCR5 • CD4


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Langerhans cells (LCs) are immature dendritic cells (DCs) located in the epidermis and in some mucosael epithelia (e.g., of the vagina, exocervix, anus, penis, and mouth) [1 , 2 ]. As antigen-presenting cells, LCs can take up and process foreign antigens at the environment interface. After an encounter with antigen, LCs migrate from the skin or mucosae to lymph nodes and become interdigitating DCs, which are mature DCs that strongly stimulate lymph node T cells [3 , 4 ]. LCs are characterized by the presence of cytoplasmic organelles, the Birbeck granules, and they express CD1a molecules on their surfaces. Cutaneous and mucosael LCs are considered functionally and structurally identical [5 , 6 ]. For experimental purposes, they are usually recovered from the skin where they are more accessible than from mucosae. They can be maintained in cultures, but they are unable to multiply. After 2 days, cultured LCs mature and resemble interdigitating DCs of lymph nodes [7 ].

LCs are probably the first cells to be infected by HIV in nonbreached multistratified epithelia, leading to viral dissemination [1 , 4 , 5 , 8 , 9 ]. LCs are the only cells in the mucosael epithelium which express both CD4 molecules and chemokine receptors [3 , 10 , 11 ]. The chemokine receptors CXCR4 and CCR5 are the coreceptors most frequently used by T-cell (T)-tropic (X4) and macrophage (M)-tropic (R5) HIV strains, respectively, to enter the target cells [12 ]. It has been demonstrated for several years that LCs can be infected by M-tropic strains of HIV-1 through CCR5 [13 ]. Until now, however, CXCR4 was found only in the cytoplasm of fresh LCs but not on their surfaces [14 ]. In contrast, mature DCs express both CCR5 and CXCR4 molecules on their surfaces. Furthermore, mature DCs can transmit M- and T-tropic HIV isolates to T cells [15 ], although HIV is blocked into DCs at an early stage of reverse transcription [10 ]. The expression of CXCR4 on the surface of CD34+ DC progenitors has been described recently [16 ]; these cells are sensitive to T-tropic HIV infection [16 ]. Considering all these data, we hypothesized that the CXCR4 receptor could also be expressed on surface membranes of freshly isolated LCs.

The expression of chemokine receptors can be modulated by several cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-10. GM-CSF can be synthesized by DCs in an autocrine pathway, and GM-CSF leads to DC maturation. It is interesting that this cytokine is known to activate HIV replication [17 ]. TNF-{alpha} is involved in LC migration [18 ] and in activation of HIV replication [17 ]. IL-10 has been shown to increase CCR5 expression and HIV infection in human monocytes [19 ]. We thus aimed to determine the effects of these cytokines on fresh LCs.

The objective of this study was to assess the presence of CXCR4 on the surface of freshly isolated cutaneous LCs and to determine the functionality of this HIV coreceptor. To optimize the infection process, we managed to obtain optimal expression of CXCR4. The density of CXCR4, CCR5, and CD4 receptor expression on the surface of LCs was measured soon after cell isolation and after culture, in the presence or absence of GM-CSF, TNF-{alpha}, and IL-10. We demonstrated that (1) CXCR4 could be expressed on the surface of 9% of fresh LCs, along with CD4 and CCR5; (2) the percentage of LCs expressing CXCR4 and CCR5 increased significantly during maturation over time (from 9% at day 0 to 55% at day 1); (3) the addition of GM-CSF but not of TNF-{alpha} and IL-10 increased the surface expression of both CXCR4 and CCR5 on LCs. We next demonstrated that CXCR4 was functional and permitted HIV entry in fresh LCs. We measured p24 production when LCs were infected with LAI, a T-tropic HIV strain, and when they were cocultured with SupT1 cells. These LCs contained proviral DNA even in the absence of coculture with SupT1 cells. These results were confirmed structurally by electron microscopy. Finally, we demonstrated that addition of recombinant SDF-1 or azidothymidine (AZT) in DCs exposed to HIV infection prevented proviral DNA integration in these LCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of LCs
Human epidermal cell suspensions were obtained from normal human skin removed during plastic surgery. Skin samples were freed of fatty tissue and split-cut with a keratome set. Skin slices were then incubated with 0.05% trypsin for 1 h at 37°C (Difco Laboratories, Detroit, MI); after this step, epidermis was separated from dermis with fine forceps. Epidermal fragments were pooled in Hanks’ balanced saline solution (Gibco, Cergy-Pontoise, France) supplemented with 10% fetal calf serum. A single epidermal cell suspension was obtained by repeated pipetting of the epidermal sheets and filtration through sterile gauze. Enrichment and purification of LCs from epidermal cell suspensions were achieved by three consecutive gradient centrifugations using lymphocyte separation medium (LSM) (Lymphoprep; Flobio SA, Courbevoie, France) for 20 min at 400 g as previously described [20 ]. The first gradient centrifugation was performed using undiluted LSM. The other two enrichment steps were done using LSM previously diluted with distilled water (3.6 mL of LSM to 1.4 mL of distilled water). LCs were recovered at the interface and were then washed and enumerated.

Cultures of LCs
LCs were resuspended in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum, 1 mL of L-glutamine (Life Technologies), and antibiotics (penicillin and streptomycin). LCs were cultured for 2 days at 106 cells/mL in 24-well culture plates (Falcon; Becton-Dickinson, Lincoln Park, NJ) with or without human recombinant cytokines at various concentrations (GM-CSF, TNF-{alpha}, or IL-10) (R&D, Minneapolis, MN).

Qualitative and quantitative immunofluorescence assay
LCs (105 cells/marker) were resuspended in PBS with 0.1% bovine serum albumin. Monoclonal antibodies (mAbs) used to label the LC suspensions were as follows: anti-CD83 (Beckman-Coulter, Margency, France), anti-CD40, anti-CD80, anti-CD86 (PharMingen, San Diego, CA), anti-human leukocyte antigen (-HLA) class II, anti-CD3, anti-CD4 (Dako, Trappes, France), anti-CXCR4, anti-CCR5 (R&D, Abingdon, United Kingdom) or isotypic control; mAbs were used under saturating conditions as described elsewhere [20 ]. Fluorescein isothiocyanate-conjugated goat anti-mouse (FITC-GAM) F(ab')2 antibody fractions were added, and free binding sites of FITC-GAM were saturated for 20 min with an irrelevant mouse immunoglobulin G2a mAb. After three washes, direct-immunofluorescence staining was performed using a phosphatidylethanolamine-conjugated anti-CD1a mAb (Dako, Trappes, France). Pellets were then washed and fixed in 100 mL of PBS containing 0.1% paraformaldehyde. Flow-cytometry analysis was performed using a FACSTAR Plus cell sorter (Becton-Dickinson, San Jose, CA).

Populations of LCs were identified according to forward and the side scatters. Quantification of cell surface molecules was performed with a quantitative immunofluorescence indirect assay (QIFIKIT®; Biocytex, Marseilles, France) as previously described [21 , 22 ]. This assay was based on the linear relationship observed between mean fluorescence intensity (MFI) and cell-bound mAb molecule numbers. Briefly, nonfluorescent plastic beads were used as fluorescence standards. They were noncovalently coated with increasing amounts of anti-CD5 mAb. Before being used, these beads were incubated for 45 min with 100 µL of a saturating amount of FITC-GAM.

Anti-CD4, anti-CXCR4, and anti-CCR5 mAbs were used under saturating conditions for 30 min at 4°C in an indirect immunofluorescence assay. After washing, FITC-GAM antibody was added, and the cells were incubated for additional 45 min. In each case, standard beads were stained, fixed, and analyzed under the same conditions and processed in parallel with the purified LCs.

Under saturating conditions, mAbs bound to cell surface antigens monovalently. For each experiment, 5,000 events were acquired. The number of antigenic sites per cell corresponded to the number of bound mAb molecules, thus defining the antibody-binding capacity. A standard regression line was calculated between MFI values, expressed as arbitrary units, and the number of cell-bound mAb molecules. The negative control was subtracted to obtain the actual data, and the corrected MFI (MFIc) was calculated for each cell and bead suspension assay and converted into so-called antigen density (i.e., the mean number of molecules expressed on the cell surface), using the following formula: MFIc = MFIexp - MFIneg, where MFIexp is the MFI for a population of interest and MFIneg is the MFI for an isotypic-matched control.

Infection of Langerhans cells
LAI T-tropic HIV viruses were recovered from SupT1 cell culture supernatants. Supernatants were cleared by centrifugation, filtered through 0.2-µm-pore-sized filters, titrated on SupT1 cells by endpoint titration, and stored at -70°C until use. LCs were incubated with LAI (tissue culture infectious dose, 20,000/mL) for 2 h and then washed three times to remove cell-free viruses. HIV-pulsed LCs were seeded into flat-bottomed 96-well plates (Costar, Cambridge, MA) at 5 x 104 cells/well in 100µL. HIV-1 pulsed LCs and SupT1 T cells (10 x105 cells/well in 100 µL) in cocultures were incubated at 37°C for 7 days. Medium was exchanged on day 4. In three experiments, LCs were preincubated for 1 h in the presence of recombinant SDF-1{alpha} (R&D Systems) or AZT (Sigma, St. Quentin Fallavier, France) before being incubated with LAI for 2 h to block interactions between HIV-1 and CXCR4 [23 ]. The HIV-1p24 protein production in culture supernatant was assessed by specific ELISA.

Electron microscopy
LCs were cultured in the presence of the LAI HIV-1 strain and cocultured with SupT1 cells as described above. Ultrastructural microscopic studies were performed at days 1 and 3 postinfection. Cells were fixed in 2% glutaraldehyde in cacodylate buffer and processed for transmission electron microscopy. Ultra-thin sections were stained with lead citrate and uranyl acetate and examined under a Jeol 1200 EX electron microscope at 80 kV of accelerating voltage (performed at the Centre de Microscopie Electronique Applique à la Biologie et à la Geologie, University of Lyon, France).

Detection of HIV-1 proviral DNA by PCR
In certain experiments, HIV-pulsed LCs were incubated for 2 h with viruses, then washed three times to remove unbound viruses, and allowed to incubate for an additional 22 h. After this period, LCs were washed, pelleted, and frozen at -70°C. After thawing, DNA was extracted by the Amplicor® DNA whole blood kit (Roche Diagnostics, Meylan, France) according to the manufacturer’s instructions. DNA was then amplified by a previously described nested-PCR protocol [24 ]. This procedure permits the amplification of a 640-bp fragment located in the reverse transcription (RT) region of the genome. The first PCR round used the following primers: RT9 (upstream; 5'-GTA CAG TAT TAG TAG GAC CTA CAC CTG TC-3') and RT12 (downstream; 5'-ATC AGG ATG GAG TTC ATA ACC CAT CCA-3'). The second PCR round used the following primers: RT1 (upstream; 5'-CCA AAA GTT AAA CAA TGG3') and RT4 (downstream; 5'-AGT TCA TAA CCC ATC CAA AC3'). For the first and the second PCR runs, amplification steps were performed with a Perkin-Elmer 9600 thermocycler as follows: 35 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and elongation at 72°C for 30 s. After the second runs, the PCR products were tested by electrophoresis at 100 V in 2% agarose gels and visualized under UV light after contact with ethidium bromide. To check that the LAI virus detected by DNA PCR in LCs did not represent residual cellular DNA from infected SupT1 cells that were used to prepare viral stocks, an aliquot of 200µL of viral stock was tested by PCR. No viral DNA could be found.

Statistical analysis
Results are expressed as means ± SD. Results were analyzed using the analysis of variance with two factors. Student’s t test was used to determine whether cytokine receptor expression was significantly modulated by the treatments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of CXCR4, CCR5, and CD4 molecules on fresh and cultured Langerhans cell membranes
LCs were recovered from epidermal cell suspensions with high yields of purification in all experiments performed (n=10). Initial epidermal cell suspensions routinely contained 1–5% LCs, whereas enriched suspensions contained 30–50% LCs after the second gradient centrifugation step and then 75–95% LCs after the third gradient centrifugation step (data not shown). Surface CD3 antigen or collagen was not observed after immunostaining, indicating that T lymphocytes or fibroblasts were not present in final cell preparations. Using flow cytometry, LCs were identified by the detection of CD1a molecules [25 ]. Fresh LCs expressed CD4, CXCR4, CCR5, CD40, and HLA class II. They did not express CD83, thus were assumed to represent immature DCs (Fig. 1 ). The number of CXCR4, CCR5, and CD4 receptors per cell was quantified by immunofluorescence (QIFIKIT®) assays.



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  		Figure 1. Surface phenotypes of fresh LCs isolated from normal human skin and studied by mAb labeling and flow-cytometric analysis. (A) Morphological gate for exclusion of debris and identification of LCs. All cytograms were conditioned on the morphological gate (expression of CD1a up to 95%) for a positive representative experiment. LCs were double stained with the following mAbs: anti-CD4 (IgG1) (C), anti-CXCR4 (IgG2a) (D), anti-CCR5 (IgG2b) (E), anti-CD40 (IgG1) (F), anti-HLA class II (G), or anti-CD83 (H). The LCs were then revealed by FITC conjugation and with rhodamine-phycoerythrin anti-CD1a. Controls were performed with antibody isotypic control revealed by FITC-GAM and rhodamine-phycoerythrin control (B).

 
CXCR4 molecules were expressed on the surface of fresh LCs in 7 of 10 skin samples, but the percentage of stained cells was low (9±9%) (Fig. 2 ). However, when expressed, the CXCR4 molecules could be clearly detected and quantified (120,598±73,539/cell). Permeabilization of LC membranes demonstrated that CXCR4 was expressed more intensively in the cytoplasm than on the surface of the tested LCs (data not shown). Furthermore, in all 10 experiments, a low percentage of fresh LCs expressed CCR5 molecules (16±11%) and a high percentage of LCs expressed CD4 molecules (95±8%) (Fig. 2) . The number of CCR5 molecules expressed on each cell was high but quite variable (91,441±82,424/cell) whereas the number of CD4 molecules was low (5,177±2,781/cell).



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  		Figure 2. Percentages (means ± SD) of LCs expressing CXCR4, CCR5, and CD4 molecules after purification and culture (10 experiments). The percentages of LCs expressing CXCR4 and CCR5 significantly increased at day 1 (P<0.01, and P=0.01, respectively). d0, day zero (fresh cells); d1, day 1 (after 24 h of culture); d2, day 2 (after 48 h of culture).

 
We also determined the kinetics of CXCR4, CCR5, and CD4 expression on LCs during culture. The percentages of LCs expressing CXCR4 and CCR5 increased significantly during culture, from 9 ± 9% to 54.9 ± 21% at day 1 and 54.8 ± 19% at day 2 (CXCR4) (P<0.01) and from 16 ± 11% to 24.6 ± 13% at day 1 and 23.7 ± 15% at day 2 (CCR5), (P=0.01) (Fig. 2) . In contrast, no change in CD4 expression was seen after 1 or 2 days of culture.

There was no significant variation of CXCR4 density on LC surfaces (99,433±91,534/cell at day 0 vs. 71,297±20,422/cells at day 1 and 98,276±38,121/cell at day 2). CCR5 expression did not change significantly (101,138±91,214/cell at day 0; 62,160±28,578/cell at day 1; and 79,266±28,893/cell at day 2). CD4 expression was 4,754 ± 3,526/cell at day 0; 3,749 ± 1,752/cell at day 1; and 2,342 ± 1,781/cell at day 2. These results are not significantly different due to the large variability depending on skin samples studied.

Modulation of expression of HIV receptors and coreceptors by Langerhans cells in culture, in the presence of exogenous cytokines
To determine whether the frequency and the density of CXCR4, CCR5, and CD4 receptor expression could be modulated by factors such as cytokines in such a manner that they can affect the infection process, LCs were exposed to GM-CSF, TNF-{alpha}, and IL-10 at various concentrations.

LCs were incubated with GM-CSF (40, 200, and 1,000 U/mL) in four separate experiments. GM-CSF, at the most efficient concentration (200 U/mL), strongly inhibited the percentage of LC expressing CCR5 after 1 day (76±6%) and 2 days (62±11%) compared with LCs cultured without cytokine (P<0.01) (Table 1 ). No effect of this cytokine was observed on CXCR4, whereas it led to a twofold increase of CD4 expression at day 2 (Table 2 ). At this concentration (200 U/mL), GM-CSF moderately though significantly increased the number of CXCR4s per cell after 1 day (115±7%) (P<0.05) and of CCR5 after 2 days (126±11%) (P<0.05) (Table 2) . Of note, GM-CSF at 40 U/mL had minimal effect and at higher concentrations, such as 1,000 U/mL, did not significantly affect CXCR4 and CCR5 expression.


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  		Table 1. Percentage of Cultured LCs Expressing CXCR4, CCR5, and CD4 in the Presence of Various Cytokines

 

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  		Table 2. Number of CXCR4, CCR5, and CD4 Molecules Expressed on LCs Cultured with Various Cytokines

 
TNF-{alpha} was used at 20, 100, and 500 U/mL in four separate experiments. A 500-U/mL concentration of TNF-{alpha} led to a significant decrease in the percentage of cells expressing CCR5 after 1 day (71±16%) (P<0.05) but did not significantly change the percentage of cells expressing CXCR4 and CD4 (Table 1) . TNF-{alpha} did not significantly affect the mean numbers of CXCR4 and CCR5 molecules expressed per cell (Table 2) . TNF-{alpha} used at lower concentrations (20 or 100 U/mL) did not significantly affect CXCR4 and CCR5 expression.

IL-10 was used at concentrations of 0.1, 1, and 10 ng/mL in five separate experiments. IL-10 affected neither the percentage of cells expressing CXCR4, CCR5, and CD4 (Table 1) nor the density of CXCR4 and CD4 molecules per cell (Table 2) . However, in 2-day cultures, 1 ng/mL of IL-10 significantly increased the expression of CCR5 (113±7%) (P<0.05), whereas 10 ng/mL of IL-10 decreased this expression (87±4%) (P<0.02).

These data showed that GM-CSF and to a lesser extent TNF-{alpha} but not IL-10, were capable of affecting the density of CXCR4 and CCR5 on the LC surface.

Infection of Langerhans cells by HIV-1 T-tropic strain
Fresh LCs exposed to GM-CSF at an optimal concentration (200 U/mL) for CXCR4 expression were infected with LAI HIV-1 strain. Productive infection was determined by the capacity of HIV-pulsed LCs to transmit infection to SupT1 cells in coculture experiments. Infection was deduced from the production of p24. Mean p24 production measured in coculture supernatant was approximately 30 ng/mL. The addition of SDF-1, a natural ligand of CXCR4 and a chemokine known to strongly inhibit infection by T-tropic HIV-1 strains [26 ], was added to LCs in culture at increasing concentrations (1, 10, and 100 ng/mL), along with infecting LAI HIV-1 strain. SDF-1, at the various concentrations used, induced a significant decrease in production of p24 (by 81, 74, and 49%, respectively) (Fig. 3 ). These data evidenced a significant blocking effect of infectivity through the use of CXCR4.



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  		Figure 3. Inhibition of LC infection with HIV-1 LAI by SDF-1{alpha}. LCs were incubated with HIV LAI strain virus with increasing concentrations of SDF-1{alpha} and then cocultured with SupT1 cells. The p24 production in four different experiments was expressed as percentages (means ± SD) in comparison with SDF-1-nontreated cells.

 
To demonstrate that LCs were effectively infected with LAI strain viruses, integrated proviral DNA presence in LCs was measured using nested PCR. As shown in Figure 4 , the presence of a specific band could be detected in LCs after incubation with the virus. Furthermore, when LCs were incubated with SDF-1 or with AZT prior to HIV infection, no proviral integration could be detected (Fig. 4) . By electron microscopy, no viral excretion was noticed from LCs. When uninfected SupT1 cells were added to virus-pulsed LCs, clustering and syncytia formation of SupT1 cells were observed after 3 days in cocultures (Fig. 5A and B ). These data clearly demonstrated that CXCR4 is involved in T-tropic HIV virus entry in fresh, immature LCs and that SDF-1 can successfully block this entry.



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  		Figure 4. Inhibition of LC infection with HIV-1 LAI by SDF-1 or by AZT. LCs were incubated with HIV-1 LAI strain virus with or without SDF-1 or AZT. LC infection was demonstrated by the presence of HIV-1 LAI proviral DNA, using nested PCR. Lane 1, negative control of the first round of PCR; lane 2, Negative control of the second PCR; lane 3, HIV-1 LAI without cells; lane 4, LCs with LAI at 4°C; lane 5, LCs incubated with HIV-1 LAI without SDF-1; lane 6, LCs incubated with HIV-1 LAI in the presence of SDF-1; lane 7, LCs incubated with HIV-1 LAI in the presence of AZT; lane 8, LCs without HIV-1 LAI; lane 9, SupT1 incubated with HIV-1 LAI; lane 10, SupT1 incubated with HIV-1 LAI in the presence of SDF-1; lane 11, SupT1 incubated with HIV-1 LAI in the presence of AZT; lane M, molecular size marker (100-bp ladder).

 


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  		Figure 5. Transmission electron microscope analysis of LC-SupT1 cell cocultures on day 3. When HIV-1 LAI-treated LCs were cocultured with SupT1 cells for 3 days, they were capable of inducing clustering (A) and syncytium formation (B) of the SupT1 cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
After sexual intercourse, cell-free or cell-associated HIV particles which are present in secretions can penetrate and cross mucosae by transcytosis in monostratified epithelia such as that of the rectum [27 ]. In contrast, in pluristratified epithelia such as that of the vagina, exocervix, penis, and eventually mouth, LCs seem to be the main target for HIV in the absence of mucosal microeffraction [28 ]. LCs are most frequently infected by M-tropic HIV-1 strains through CCR5, because these HIV-1 strains are predominant during primoinfection. After encounter with the virus, LCs migrate through the chorion to the lymph nodes and can transmit HIV to T cells [4 ]. This mechanism of HIV transmission is supported by studies on vaginal and oral inoculations in simian immunodeficiency virus experimental infections in monkeys [29 , 30 ]. In vivo, LCs undergo specific morphologic and phenotypic changes during migration [7 ]. We considered that LC maturation could modify HIV infection and/or HIV tropism by influencing the expression of HIV receptors on plasmic membranes. In this study, we thus analyzed the differential expression of HIV receptors/coreceptors on human LCs freshly isolated from skin biopsies and during short-term cultures assumed to mimic the in vivo cell migration. To increase the expression of HIV receptors on LC surfaces and subsequently to optimize the infection process, we tested for the effects of various exogenous cytokines that have been reported to modulate HIV infection [17 , 19 ]. We were able to demonstrate that fresh LCs could express membrane CXCR4. We next investigated whether the CXCR4 coreceptor was functional. To the best of our knowledge, this observation is the first report on the expression of CXCR4 on freshly isolated LCs.

In the experimental conditions used, the level of LC purification was in a mean range of 85%. They were contaminating neither CD3+ T cells nor fibroblasts in the cell suspensions. There were no mature DCs in these suspensions either. A decrease in CD1a and an increase in HLA-DR expression during cell cultures confirmed the DCs differentiation in vitro (data not shown), and after 2 days of culture, the initial LCs resembled interdigitated DCs [7 ].

The present study was thus performed on highly enriched LC fractions. It confirmed that fresh LCs expressed the CD4 receptor and the M-tropic HIV-1 coreceptor CCR5. We report herein that fresh LCs also expressed the T-tropic HIV-1 coreceptor CXCR4 on the plasmic membrane in approximately 9% of freshly isolated cells from 70% of the skin biopsies studied. We next confirmed that CXCR4 molecules were present in freshly isolated LCs but were mainly located in the intracytoplasmic area [6 , 14 ] possibly because they are rapidly internalized [31 ]. In a previous study, Zoeteweij et al. showed that, after 2 days, cultured LCs expressed significant levels of CXCR4 and CCR5; however, these authors did not study CXCR4 expression on freshly isolated LCs [32 ]. In another study, Zaitseva et al. were unable to demonstrate the presence of CXCR4 on fresh LC surface membranes [14 ]. This discrepancy could be explained by a different methodological approach in cell preparation and antigen detection. The CXCR4 expression by fresh LCs might have been altered by the technique (suction blister) used by Zaitseva et al. for epidermis isolation. Furthermore, CXCR4s as well as other LC surface molecules were very sensitive to enzymatic cleavage by the relatively high doses of trypsin used by Zaitseva et al. (0.25% vs. 0.05% in our study) [20 ]. It is noteworthy that these studies were performed using various anti-CXCR4 antibodies, which may also account for certain discrepancies. Our results, however, are in agreement with the majority of previous studies demonstrating (1) that CXCR4 is expressed on CD34+ progenitor cells [16 , 33 ], on immature DCs [34 , 35 ]—which correspond to lower differentiation stages of DCs [13 , 35 36 37 ], and on mature DCs [14 , 32 ], and (2) that LCs can be infected by T-tropic HIV strains [6 , 13 , 35 36 37 ].

In contrast to the low expression of CXCR4 detected on fresh LCs, CCR5 was detected at a mean range of 16% and CD4 at a range of 95% on LC surfaces immediately after the cell purification in all skin samples studied, as expected [14 ].

HIV receptor expression was analyzed on LCs during their maturation in short-term cultures. Mature DCs resembling interdigitating DCs expressed CXCR4, CCR5, and CD4 molecules as expected [15 ]. The expression of CXCR4 and CCR5 on the LC surface increased during culture (and approximately 55% of LCs expressed CXCR4 and 25% expressed CCR5 on day 2). The increase in cellular receptor expression was rapid, probably implicating an important protein turnover and/or a very efficient plasmic membrane delivery mechanism. Our results are in agreement with those from previous studies challenging LCs after maturation in culture [14 , 32 ] or DCs derived from blood-circulating progenitors [38 ], with T-tropic HIV strains [10 , 14 , 15 , 32 , 39 ].

The effects of exogenous cytokines on HIV receptor/coreceptor expression were further evaluated since they can play an important role in HIV infection. We found that the addition of GM-CSF in culture increased the density of CXCR4 as well as CCR5 on LC surfaces and decreased the percentage of LCs expressing CCR5 but not the percentage of LCs expressing CXCR4. In previous studies, GM-CSF was reported to decrease CCR5 and CXCR4 mRNA expression in macrophages, which was correlated with a decreased ability to support HIV entry [40 ]. Furthermore, GM-CSF did not appear to affect the replication in vitro of M-tropic HIV-1 strains in LCs [39 ]. It is not known whether GM-CSF directly affects CCR5 expression or whether GM-CSF induces cytokine or chemokine production which in turn regulates the chemokine receptor surface levels. Further studies are required to clarify this point.

TNF-{alpha} has been reported to be a potential agent for increasing HIV replication in LCs [39 ]. We found that it decreased the percentage but not the cell surface receptor density of LCs expressing CCR5. No effect was observed on CD4 or CXCR4 expression. These results suggest that the effect of TNF-{alpha} observed in vitro on HIV infection is not related to a modification of receptor expression but could be related to an effect on HIV intracellular transport or replication mechanisms [17 ]. However, TNF-{alpha} could also be involved in stimulating LC migration across the epithelium [18 ].

We found that the addition of IL-10 increased CCR5 density on LC surfaces after 2 days of culture when used at a low concentration (1 ng/mL) but that it decreased the CCR5 density when used at a high concentration (10 ng/mL). Similar data were reported on monocytes [19 ]. IL-10 is reported to have dual effects on HIV entry into monocytes: (1) it inhibits HIV replication in monocytes by inhibiting cell differentiation [41 ]; or (2) it stimulates infection with M-tropic HIV-1, possibly by enhancing viral entry through the use of CCR5 [19 ].

Taken together, these results indicate that HIV coreceptor expression could be specifically modulated by cytokines and that these cytokines were involved in LC infection by HIV. Recent studies have demonstrated the influence of HIV receptor cell surface density on cell infectivity. It seems that sexual transmission of HIV M-tropic (R5) isolates depends on the cellular density of CCR5 and CD4 [42 ]. Although it is well established that M-tropic viruses are the most frequently involved viruses in sexual transmission, it has been recently demonstrated that T-tropic strains can be found in epithelia after sexual contact in humans [43 ] and in macaques [11 ]. Infections by primary T-cell-tropic isolates would be highly dependent on cellular CD4 levels, whereas the M-tropic isolates appeared more able to infect cells with low amounts of CD4 [43 ]. The relatively low density of CD4 molecules that we observed on LC surfaces might also contribute to the sexual transmission of HIV R5 virus isolates across mucosae in preference to CXCR4 (X4) viruses. The independent modulation of CCR5 and CXCR4 coreceptors provides new approaches for studying the cellular density needed for HIV infection and for studying HIV tropism. Studies are underway in our laboratory to further analyze these issues.

To test the functionality of the CXCR4 coreceptor, we studied the infection of fresh LCs with the LAI T-tropic HIV strain in the presence of GM-CSF. After incubation of LCs with the virus, cocultures with human target T cells supported productive infection, but LCs alone were not susceptible to virus replication and did not produce p24. When LCs were treated with SDF-1 before coculture with SupT1 cells, the p24 production by cocultured cells was dramatically reduced. Suppression of CXCR4 expression was rapidly reversed on removal of SDF-1, suggesting that such effects are mediated by internalization into endosomal compartments and subsequent recirculation to the cell surface [31 ]. We demonstrated that CXCR4 coreceptor was functional on fresh-LC surfaces because proviral DNA could be detected in LCs and such proviral production could be blocked by preincubation of LCs in the presence of SDF-1{alpha} or AZT. AZT was added to block RT in the DCs [44 , 45 ]. A 2-h incubation of HIV-1 LAI with epidermal LCs rendered the cells able to transmit the virus to SupT1 cells, which was confirmed by the microscopic observation of clustering and syncytium formation with SupT1 cells. It is known that DC–T-cell conjugates but not purified DCs or T cells were productively infected by HIV-1 [5 ]. Using PCR, Pope et al. demonstrated that free DCs and T cells had less than 100 copies of HIV-1 DNA per 5 x 104 cells [44 ]. The recently described DC-specific, intercellular adhesion molecule 3-grabbing, nonintegrin ("DC-SIGN") receptor is not expressed on the surface of LCs within mucosal epithelia [46 ], which makes this pathway very unlikely for HIV-1 transmission to permissive T cells.

Although M-tropic HIV-1 strains are implicated in approximately 90% of sexual transmissions of the virus, T-tropic HIV strain transmission using the CXCR4 pathway is thus possible [47 ]. CD34+ cells cultured for 12 days with GM-CSF, TNF-{alpha}, SCF, and IL-4 can differentiate into DCs, that is at a stage of differentiation very similar to that of LCs, and can be infected by X4 strains of HIV [37 ]. Individuals with the CCR5{delta}32/CCR5{delta}32 genotype, leading to an inoperative CCR5 molecule, can be infected with HIV [48 ]. As previously reported, mature DCs with the CCR5{delta}32 point mutation can transmit a T-tropic virus strain to T cells, suggesting that they could use an alternate pathway, which probably involves CXCR4 [15 ].

The data reported herein thus show that fresh LCs, which are at a stage resembling mucosal LCs potentially exposed to HIV infection after sexual intercourse, could functionally express the CXCR4 HIV-1 coreceptor along with the already described CCR5 coreceptor.


   ACKNOWLEDGEMENTS
 
This work has been supported by the French "Agence Nationale de Recherche sur le SIDA (ANRS)" and by the Région Rhône-Alpes. It has been supported by a grant from the ANRS. We are very grateful to Drs. F. Souliard (Clinique Jomayère, Saint Etienne) and T. Lefort (Hospital Bellevue, Saint Etienne) for providing skin sample. We acknowledge the assistance of Drs. V. Atrache (Perth, Australia) and N. Vincent, E. Malvoisin, and F. Grattard (GIMAP, Saint-Etienne). We thank Drs. O. Garraud and S. Riffard (GIMAP, Saint-Etienne) for comments and critical review of the manuscript.

Received August 9, 1999; revised April 2, 2001; accepted April 5, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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