Departamento de Genética y Biología Molecular, Centro de Investigación y Estudios Avanzados-IPN, Av. IPN No. 2508, Col. San Pedro Zacatenco CP 07360, Mexico DF, Mexico1
Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico DF, Mexico2
Author for correspondence: Bertha Moreno-Altamirano. Fax +1 525 747 7100. e-mail bertha{at}lambda.gene.cinvestav.mx
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Abstract |
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Introduction |
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DEN causes an estimated 100 million new cases of DF, 500000 cases of DHF and 25000 deaths annually (Gubler, 1998 ). Moreover, 2·5 billion people are at risk (Monath, 1994
).
Studies of pathological specimens from patients with DHF suggest that many tissues may be involved, as viral antigens are expressed in the liver, lymph nodes, spleen and bone marrow (Couvelard et al., 1999 ; Kurane et al., 1994
; Mangada & Igarashi, 1998
; Rosen et al., 1999
).
Although monocytes and macrophages have been reported to display DEN antigens in pathological specimens from patients with DHF (Halstead, 1989 ), it is still a matter of speculation as to which tissue cells are the natural targets in which DEN reside and produce virus particles, since, in addition to monocytes and macrophages, T lymphocytes, dendritic cells, hepatocytes, endothelial cells, epithelial cells and fibroblasts have also been reported as potential hosts for DEN (Avirutnan et al., 1998
; Bonner & OSullivan, 1998
; Despres et al., 1998
; Ho et al., 2001
; Kurane et al., 1990
, 1992
; Leitmeyer et al., 1999
; Marianneau et al., 1997
; Mentor & Kurane, 1997
; Wu et al., 2000
). In primary DEN infections, DEN enters the target cell after the envelope protein E attaches itself to an as yet ill-characterized receptor(s) on the cell membrane. In secondary infections, viruses may enter the cells through a primary receptor but may also form immune complexes with pre-existing, non-neutralizing antibodies and interact with alternative receptors, such as Fc
receptors I and II (Daughaday et al., 1981
; Littaua et al., 1990
).
Considering that observations on anatomical sites of DEN replication in humans are compatible with the possibility that cells of the monocyte-macrophage lineage in the lymphoid organs, lung and liver serve as significant targets of infection (Scott et al., 1980 ; Hall et al., 1991
), it seemed worthwhile to try to identify the putative receptors for DEN-2 in human macrophages.
The human promonocytic U-937 cell line has frequently been used as a model to study macrophage function (Sundstrom & Nilsson, 1976 ; Larrick et al., 1980
). Accordingly, along with the establishment of human peripheral blood monocyte-macrophage primary cultures for our study, undifferentiated as well as phorbol myristate acetate (PMA)-differentiated U-937 cells were analysed for their ability to support DEN-2 binding.
Here we provide evidence that four proteins of human monocyte-derived macrophages, with molecular masses of 27, 45, 67 and 87 kDa (27K, 45K, 67K and 87K proteins), may serve as receptors for DEN-2. On the other hand, three of the proteins (45K, 67K and 87K) from PMA-differentiated U-937 cells were retained by a DEN-2-bound Sepharose 4B column, but only the 45K and 67K proteins were recognized by DEN-2 in virus overlay protein-binding (VOPB) assays. Two proteins (67K and 87K) from undifferentiated U-937 cells were retained by the DEN-2-bound Sepharose 4B column but neither protein interacted with DEN-2 in VOPB assays. These differences in DEN-2 membrane protein interaction may provide an explanation of why macrophages but not undifferentiated U-937 cells are susceptible to primary infection with DEN (OSullivan & Killen, 1994 ).
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Methods |
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Cells.
Monocyte-derived macrophages were obtained from the peripheral blood from healthy donors. Leukocyte-enriched plasma was centrifuged over FicollHypaque (Pharmacia). Mononuclear cells were washed, resuspended in Dulbeccos modified Eagles medium (DMEM) (Gibco) and plated into plastic Petri dishes (Costar). After 2 h of incubation at 37 °C in a 5% CO2 atmosphere, non-adherent cells were removed. Adherent cells were further incubated for 35 days to allow monocytes to differentiate into macrophages. The promonocytic cell line U-937 (Sundstrom & Nilsson, 1976 ) was maintained in DMEM supplemented with 5% foetal calf serum (FCS) (Gibco) at 37 °C in a 5% CO2 atmosphere. For experiments, U-937 cells were subcultured in two groups in 100 mm Petri dishes. The first group of cells were cultured for 3 days in medium alone. The second group of cells were stimulated for 3 days with 100 nM PMA (Sigma) in order to induce their differentiation to the monocytic stage (Ralph et al., 1982
; Moretta et al., 1977
).
Immunofluorescence.
In order to assess the binding of DEN-2 to the cell membrane of the cells under study, suspensions of undifferentiated U-937 cells, PMA-differentiated U-937 cells and human monocyte-derived macrophages cultured on coverslips were exposed to DEN-2 in minimal essential medium (MEM), pH 6·8, without FCS at 37 °C for 45 min. After this time, cells were washed with PBS and fixed with cold acetone for 10 min. Cells were then blocked with 3% BSA (Sigma) in PBS for 30 min at room temperature, after which cells were incubated for 2 h at room temperature in the presence of the anti-DEN envelope (E) protein monoclonal antibody (mAb) 2C5.1 (kindly provided by Dr A. Falconar, University of Oxford, UK). After washing three times with PBS, FITC-labelled goat anti-mouse IgG (IgGFITC) (Sigma) was added to both cells in suspension and cells in monolayers. Cells were further incubated and, after additional washing steps, mounted in Vectashield (Vector) on glass slides and observed under a fluorescence microscope (Zeiss).
Membrane protein extraction and iodination.
After culture, monocyte-derived macrophages, undifferentiated U-937 cells and PMA-differentiated U-937 cells were sonicated (Vibra cell) by giving four bursts of 5 s each in the presence of protease inhibitors (1 mM PMSF, 20 mM aprotinin, 20 mM leupeptin and 2 mM EDTA) (Sigma). Cell membranes were isolated by layering sonicated cells onto a 41% sucrose homogenizing buffer (3:1, v/v) and centrifuged at 95000 g for 1 h. The cell membrane-containing white interphase was collected and washed twice with homogenization buffer (10 mM sodium phosphate, 1 M MgCl2, 30 mM NaCl, 1 mM DTT, 0·5 mM PMSF and 0·05 µg/ml DNase, pH 7·4). The membrane proteins were then solubilized with Triton X-114 in the presence of protease inhibitors and iodinated by the lactoperoxidase method described by Morrison (1980) .
Affinity chromatography, SDSPAGE and autoradiography.
Solubilized and radio-labelled cell membrane proteins were incubated with agitation for 30 min at 37 °C with a DEN-2-bound Sepharose 4B column. After extensive washing with 25 mM TrisHCl, pH 7·2, the retained proteins were eluted with 0·5 M NaCl for 1 h with constant agitation. Protein was precipitated with ice-cold, absolute ethanol and suspended in lysis buffer containing 1% SDS, 1% Triton X-114 and a cocktail of protease inhibitors. Radioactive emission was adjusted to 80000 c.p.m. and then proteins were separated by 12% SDSPAGE and observed by autoradiography.
VOPB assay.
In order to identify the cell membrane proteins involved in DEN binding, a VOPB assay was carried out. Total membrane protein (70 µg) from undifferentiated U-937 cells, PMA-differentiated U-937 cells and human monocyte-derived macrophages were loaded into separate wells of a 12% SDSpolyacrylamide gel and subjected to electrophoresis (SDSPAGE). When the separation of proteins was complete, proteins were transferred onto nitrocellulose (Millipore) using transfer buffer (48 mM Tris, 39 mM glycine and 20%, v/v, methanol) in a semidry blotting apparatus (Hoeffer). The nitrocellulose sheets were blocked with blocking buffer [3% powdered skimmed milk and 0·1% Tween-20 in PBS (PBS-T), pH 7·4] for 1 h at room temperature and then incubated with gentle rocking overnight at 4 °C with DEN-2 in PBS-T containing 1% skimmed milk. Afterwards, nitrocellulose sheets were washed three times, for 10 min each, with PBS-T at room temperature and then incubated for 2 h at room temperature with mAb 2C5.1.
Nitrocellulose sheets were washed with PBS-T and incubated with peroxidase-conjugated rabbit anti-mouse IgG (IgGPO) (Sigma). After further washing steps, the nitrocellulose sheets were developed using enhancement chemiluminescence substrate (ECL) (Amersham) and autoradiography (Kodak).
Preparation of polyclonal antibodies (pAbs) and assessment of specificity.
The macrophage membrane proteins that were retained by the DEN-2-bound Sepharose 4B column, as described above, were separated by 12% SDSPAGE. After silver staining, each of the four main protein species was excised from the gel. The polyacrylamide gel was then disrupted into very small pieces, suspended in PBS and mixed with an equal volume of Titre-Max adjuvant (CyTRx Corporation). Four groups of BALB/c mice were bled to obtain pre-immune sera and then immunized with the 27K, 45K, 67K or 87K protein (30 µg protein per mouse). After 2 weeks, the mice received a booster and, 2 weeks later, were bled to obtain immune sera. Sera were stored at -20 °C until use.
In order to assess the specificity of the pAbs, two methods were followed. First, human monocyte-derived macrophages were obtained, as described above, and plated onto coverslips in 6-well microtitre plates. Pre-immune sera as well as each of the pAbs (anti-27K, anti-45K, anti-67K and anti-87K) were then independently added to the macrophages. After 1 h of incubation at 37 °C, cells were washed three times with PBS and then incubated for 1 h in the presence of goat anti-mouse IgGFITC. After washing with PBS, cells were mounted in Vectashield and observed under a fluorescence microscope.
In addition, human macrophage cell membrane proteins were subjected to 12% SDSPAGE and transferred to nitrocellulose. Nitrocellulose was longitudinally cut and, after blocking with blocking buffer, every piece was individually tested for reactivity with each of the four mouse pAbs (anti-27K, anti-45K, anti-67K and anti-87K), as well as with the pre-immune sera, by overnight incubation in the presence of the respective sera at 4 °C, followed by washing and further incubation with rabbit anti-mouse IgGPO for 2 h at room temperature. Western blots were developed using ECL and autoradiography.
pAb-mediated infection inhibition assays.
Inhibition of DEN-2 infection with the mouse pAbs was carried out exclusively on human macrophage primary cell cultures by two different methods: flow cytometry and immunofluorescence combined with optical scanning. In both cases, monocyte-derived macrophages were obtained as described above. For flow cytometry, cells were seeded in 6-well microtitre plates and, after 1 h exposure to each of the four mouse pAbs raised against the putative receptors for DEN-2 or with the corresponding pre-immune sera (in separate wells), cells were infected with DEN-2 (excess antibody was removed prior to virus infection). Infection was allowed to proceed for 72 h, after which cells were permeabilized with 0·5% saponin (Sigma) in PBS and fixed with 4% paraformaldehyde (Sigma) in PBS. Blocking was carried out with 3% BSA in PBS for 30 min at room temperature and cells were labelled with the anti-DEN-2 non-structural 1 (NS1) protein mAb 1A12.2 (kindly provided by Dr A. Falconar) (Falconar et al., 1994 ) followed by goat anti-mouse IgGFITC. After 1 h of incubation and washing, the percentage of infected cells was assessed in a FACSCalibur flow cytometer (Becton-Dickinson) and data were analysed with the aid of the CELLQUEST software (Becton-Dickinson).
For immunofluorescence and optical scanning, macrophages were cultured, exposed to the mouse pAbs or pre-immune sera and infected with DEN-2 in Lab-Tek chamber glass slides (Nunc). After 72 h of infection, cells were intracellularly labelled with mAb 2C51 followed by washing with PBS and labelling with goat anti-mouse IgGFITC. After further washing steps, the intensity of fluorescence in each cell culture was determined under a Typhoon 8600 optical scanner (Molecular Dynamics). Fluorescence intensity (indicative of infection) was represented as fluorescent units. After optical scanning, cells were mounted in Vectashield and fluorescein-labelled cells were observed under a confocal microscope (Zeiss).
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Results |
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To further evaluate the involvement of the 27K, 45K, 67K and 87K macrophage membrane proteins in DEN-2 infection, immunofluorescence and optical scanning were applied. This method also demonstrated pAb-dependent inhibition of DEN-2 infection of human macrophages. Fig. 6(A) shows the level of expression of DEN-2 envelope protein in DEN-2-infected human macrophages (in fluorescent units) for each treatment. As can be seen, the highest level of fluorescence is in the DEN-2-infected cells not previously exposed either to pAbs or to pre-immune sera. When cells were incubated in the presence of any of the mouse pAbs (anti-27K, anti-45K, anti-67K or anti-87K), the number of fluorescent units in the cultures dropped in comparison to the cell cultures that were treated with the corresponding pre-immune sera, indicative of pAb-mediated inhibition of infection. Fig. 6
shows fluorescent cells (DEN-2 E protein expression) as observed by confocal microscopy, DEN-2-infected macrophages, macrophages treated with pre-immune sera and then infected with DEN-2 and macrophages treated with the anti-67K pAb and then infected with DEN-2. Only the immune serum was able to diminish the number of DEN-2 E protein-expressing cells. Similar results were obtained when cells were treated with the anti-27K, anti 45K and anti-87K pAbs (data not shown).
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Discussion |
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The way DEN enters the host cell still awaits better definition. It is known that during primary infection, DEN binds to an as yet ill-defined host cell membrane receptor(s) and that in the course of secondary infections, an antibody-mediated mechanism of virus entry takes place, where virusantibody complexes bind to host cell FcRs, after which the whole complex enters the cell by entocytosis (Gollins & Porterfield, 1995 ; Mady et al., 1993
). In this regard, antibody-mediated enhancement of DEN infection is a well-documented fact (Morens, 1994
; McBride & Bielefeldt-Ohmann, 2000
). Based on epidemiological and in vitro studies, the antibody-dependent enhancement of infection theory attempts to explain the pathogenesis of DHF/DSS. In studies conducted mainly in Thailand, it was observed that DHF occurred predominantly in children undergoing a second infection with a DEN serotype different from the one encountered in the first infection (reviewed by Morens, 1994
; McBride & Bielefeldt-Ohmann, 2000
). Along the same line, there is evidence that in the presence of cross-reactive but not neutralizing antibodies, cells of the macrophage lineage are more readily infected with DEN (Morens, 1994
).
Primary DEN-2 infection mechanisms are less well understood, although several putative receptors for various DEN serotypes in different cell types have been described, which could help to better understand primary infections: notably a 100 kDa protein of K-562 cells acts as a receptor for DEN-2 (Rothwell et al., 1996 ), a type of glycosaminoglycan (highly sulfated heparan sulfate) in Vero and CHO cells acts as a receptor for DEN-2 (Chen et al., 1997
), two proteins of 40 and 45 kDa in C6/36 Aedes albopictus cells act as receptors for DEN-4 (Salas-Benito & del Angel, 1997
), a trypsin-resistant 65 kDa molecule in N1E-115 (murine neuroblastoma) and SK-N-SH (human neuroblastoma) cells acts as a receptor for DEN-2 (Ramos-Castañeda et al., 1997
) and two proteins of 67 and 80 kDa in C6/36 cells act as receptors for DEN-2 (Muñoz et al., 1998
). More recently, a molecule associated with CD14 has been suggested as a receptor for DEN (Chen et al., 1999
), another report suggests a protein of 74 kDa that might participate as a co-receptor in DEN-4 infection of Vero cells (Martínez-Barragán & del Angel, 2001
).
The results presented in this study provide evidence that human monocyte-derived macrophages are able to support binding and replication of DEN-2 and that 27K, 45K, 67K and 87K cell membrane proteins could serve as DEN-2 receptors in human macrophages in primary DEN-2 infections. Results from DEN-2-bound Sepharose 4B column affinity chromatography and VOPB assays strongly suggest that these molecules are receptors for DEN-2, either as independent receptors or as subunits from a more complex receptor. Although, for unknown reasons, there is not a strict correlation between the results obtained from the two methods, i.e. membrane proteins from undifferentiated U-937 cells that were retained by the DEN-2-bound Sepharose columns were not observed in the VOPB assays. Since nitrocellulose-bound proteins are supposed to conserve their conformation and, therefore, their ability to bind their ligands, the most likely explanation for this discrepancy rests in the different sensitivity of the two methods or indeed in a lower amount of DEN-2 receptors in undifferentiated cells. The latter is supported by the fluorescent-based binding experiments (Fig. 1). In any event, from the three types of cells analysed, the human monocyte-derived macrophages were the ones that performed best in affinity chromatography and VOPB assays and also the ones that strongly supported DEN-2 infection, as assessed by immunofluorescence and flow cytometry.
Two proteins (45K and 67K) were also identified as putative DEN-2 receptors in PMA-differentiated U-937 cells. Differences in DEN-2-binding proteins between primary cultures of human monocyte-macrophages and U-937 cells, along with the fact that U-937 cells are less likely to be primarily infected with DEN-2, suggest that U-937 cells are perhaps not the best model to study primary DEN-2 infection of monocyte-macrophages.
Mouse polyclonal antisera raised against the putative receptors for DEN-2 in human monocyte-derived macrophages proved to be inhibitory for DEN-2 infection in two different methods, thus implying a role for these four proteins in virus entry to the host cell. Flow cytometry assessed the percentage of infected cells, whereas optical scanning assessed total fluorescence units.
It is interesting to note that proteins with molecular masses similar to the ones reported here have been shown to act as receptors for DEN in other cell types, particularly a 45 kDa protein in the C6/36 cell line (from A. albopictus larvae) (Salas-Benito & del Angel, 1997 ) and a 67 kDa protein also in C6/36 cells (Muñoz et al., 1998
). In addition, polyclonal antisera raised against the human monocyte-derived macrophages 67K protein recognizes a 67 kDa cell membrane protein in C6/36 cells and is able to inhibit infection of these cells with the New Guinea strain of DEN-2 (M. L. Muñoz & R. Tovar-Gallegos, unpublished observation). Thus, it seems that the receptors for DEN could be structures that are conserved, but this remains to be elucidated and protein sequencing of the different DEN-2 receptors will help to clarify this matter.
In conclusion, our results strongly suggest that four proteins (27K, 45K, 67K and 87K) participate in DEN-2 infection of human macrophages.
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Acknowledgments |
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References |
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Received 5 September 2001;
accepted 4 January 2002.