Non Fc receptor-mediated infection of human macrophages by dengue virus serotype 2

M. M. Bertha Moreno-Altamirano1, F. Javier Sánchez-García2 and M. Lourdes Muñoz1

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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Four human monocyte-derived macrophage membrane proteins, with apparent molecular masses of 27, 45, 67 and 87 kDa, were identified as possible receptors for dengue virus serotype 2 (DEN-2) (Mexican isolate 200787/1983), based on affinity chromatography, immunofluorescence, virus overlay protein-binding assays and Western blotting. Additionally, mouse polyclonal antibodies raised against each of the four proteins were capable of partially inhibiting in vitro DEN-2 infection of monocyte-macrophages, thus supporting the notion of a role for such proteins as DEN-2 receptors. Parallel studies were carried out using the human promonocytic U-937 cell line, both as undifferentiated cells and as monocyte-like phorbol myristate acetate (PMA)-differentiated cells, as target cells. Whereas interaction between DEN-2 and undifferentiated U-937 cells was almost negligible, PMA-differentiated U-937 cells were shown to harbour putative receptors (with molecular masses of 45 and 67 kDa) for DEN-2, similar to those found in human monocyte-derived macrophages. To our knowledge, this is the first report that describes putative receptors for DEN-2 in primary cultures of human macrophages.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Dengue viruses (DEN) are arthropod-borne flaviviruses that cause serious human diseases worldwide, principally in the tropical areas of Asia, Oceania, Africa and the Americas (McBride & Bielefeldt-Ohmann, 2000 ). There are four serotypes of DEN (DEN-1, -2, -3 and -4) and infection by any of them may result in either a relatively benign fever, called dengue fever (DF), or a fatal disease, such as dengue haemorrhagic fever (DHF) or dengue shock syndrome (DSS).

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 & O’Sullivan, 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{gamma} 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 (O’Sullivan & Killen, 1994 ).


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus.
DEN-2 Mexican strain 200787/1983, kindly provided by Dr B. Ruiz and Dr I. Sánchez (IIB-UNAM, Universidad Nacional Autonoma de Mexico, Mexico) (Ruiz et al., 1999 ), was expanded in Vero cells, purified as described previously (Gould & Clegg, 1991 ) and kept frozen (-70 °C) as a stock until use. The titre of the virus stock used for this study was 8x109 p.f.u./ml.

{blacksquare} Cells.
Monocyte-derived macrophages were obtained from the peripheral blood from healthy donors. Leukocyte-enriched plasma was centrifuged over Ficoll–Hypaque (Pharmacia). Mononuclear cells were washed, resuspended in Dulbecco’s modified Eagle’s 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 3–5 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 ).

{blacksquare} 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 (IgG–FITC) (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).

{blacksquare} 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) .

{blacksquare} Affinity chromatography, SDS–PAGE 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 Tris–HCl, 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% SDS–PAGE and observed by autoradiography.

{blacksquare} 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% SDS–polyacrylamide gel and subjected to electrophoresis (SDS–PAGE). 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 (IgG–PO) (Sigma). After further washing steps, the nitrocellulose sheets were developed using enhancement chemiluminescence substrate (ECL) (Amersham) and autoradiography (Kodak).

{blacksquare} 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% SDS–PAGE. 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 IgG–FITC. 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% SDS–PAGE 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 IgG–PO for 2 h at room temperature. Western blots were developed using ECL and autoradiography.

{blacksquare} 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 IgG–FITC. 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 IgG–FITC. 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).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
DEN-2 binding to the cell surface of human monocyte-derived macrophages, U-937 cells and PMA-differentiated U-937 cells
There is some controversy regarding the binding of DEN to human macrophages and to U-937 cells (O’Sullivan & Killen, 1994 ). Accordingly, we first tested whether this interaction really does take place. With the aid of DEN-specific mAbs and immunofluorescence, it was shown that DEN readily attaches to the cell surface of human monocyte-derived macrophages. In order to test if the same happens with the human promonocytic U-937 cell line, cells were both left undifferentiated or PMA-differentiated to the monocytic stage (Ralph et al., 1982 ) and then exposed to DEN-2. U-937 cell differentiation was assessed by the ability of those PMA-treated cells to bind to IgG-coated, sheep red blood cells (IgG–SRBC), indicative of FcR expression (Larrick et al., 1980 ). As expected, undifferentiated U-937 cells failed to bind to IgG–SRBC, whereas PMA-differentiated U-937 cells did (data not shown). Fig. 1 shows the typical pattern of DEN-2 binding (representative of six independent experiments) to the cells under study. Clearly the ability of both undifferentiated and PMA-differentiated U-937 cells to support DEN-2 binding is lower than that of primary cultures of human macrophages.



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Fig. 1. Binding of DEN-2 to the cell surface of human monocyte-derived macrophages, undifferentiated U-937 cells and PMA-differentiated U-937 cells. Cells were exposed to DEN-2 for 45 min. Attachment of DEN-2 to the cell surface was assessed by immunofluorescence using the anti-E protein mAb 2C5.1 followed by goat anti-mouse IgG–FITC pAbs. (A) DEN-2 binding to macrophages; (B) visible light microscope image of the same cells; (C, D) cells treated in the same way except for the DEN-2; (E, F) DEN-2 binding to PMA-differentiated U-937 cells and undifferentiated U-937 cells, respectively. Results are representative of at least six independent experiments.

 
Cell membrane proteins retained by DEN-2-bound Sepharose 4B columns
In an attempt to identify human macrophage cell surface proteins with the ability to bind to DEN-2, affinity chromatography was carried out by passing iodinated cell membrane proteins from undifferentiated U-937 cells, PMA-differentiated U-937 cells and human macrophages through a DEN-2-bound Sepharose 4B column. Fig. 2 shows a representative (out of three experiments) array of proteins that were retained by the column. Four proteins, with apparent molecular masses of 27, 45, 67 and 87 kDa, from human macrophages were retained by the column (indicative of DEN-2–cell membrane protein interaction) (Fig. 2, lane 3). A similar array was observed for PMA-differentiated U-937 cell membranes, particularly the 45K, 67K and 87K proteins (Fig. 2, lane 2). An additional protein, with an apparent molecular mass of about 56 kDa, was also retained by the column, whereas the 27K protein observed in macrophages was absent in PMA-differentiated U-937 cells. From undifferentiated U-937 cells (Fig. 2, lane 1), only a small amount of the cell membrane 67K and 87K proteins was retained by the DEN-2 column. In conclusion, PMA-differentiated U-937 cells and human macrophage primary cell cultures share three proteins, 45K, 67K and 87K, but there are some cell type-specific differences with regard to other protein species.



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Fig. 2. Cell membrane proteins retained by DEN-2-bound Sepharose 4B column. Undifferentiated U-937 cells (lane 1), PMA-differentiated cells (lane 2) and human macrophages (lane 3) cell membrane proteins were radiolabelled (125I) and passed through a DEN-2-bound Sepharose 4B column. Retained proteins were eluted and separated by 12% SDS–PAGE. Autoradiography shows the cell membrane protein species retained in the Sepharose 4B column and is representative of three experiments.

 
DEN-2 binding to cell membrane proteins
As a second approach to try to identify macrophage cell surface proteins that interact with DEN-2, cell membrane proteins from the three cell types under study were separated by electrophoresis and transferred onto nitrocellulose membranes, to which VOPB assays were performed (three independent experiments) as described in Methods. Fig. 3 shows that DEN-2 binds to the 27K, 45K, 67K and 87K cell membrane proteins from human macrophages and that the strongest DEN-2 binding was to the 67K protein. DEN-2 binds to the 67K protein and, to a lesser extent, to the 45K protein from PMA-differentiated U-937 cells. DEN-2 binding to cell membrane proteins from undifferentiated U-937 cells was below detectable levels, if any.



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Fig. 3. DEN-2 VOPB assays. Undifferentiated U-937 cells (lane 1), PMA-differentiated U-937 cells (lane 2) and human macrophage (lane 3) cell membrane proteins were separated by 12% SDS–PAGE and transferred to nitrocellulose. Nitrocellulose sheets were layered with DEN-2 and then incubated with the anti-E protein mAb 2C5.1 and rabbit anti-mouse IgG–PO pAbs. Nitrocellulose sheets were treated with ECL reagent and autoradiographed.

 
Mouse pAb-mediated inhibition of DEN-2 infection of human macrophages
Antibody specificity was assessed as described in Methods. All four pAbs (anti-27K, anti-45K, anti-67K and anti-87K) were shown to recognize human macrophage cell membranes by immunofluorescence, whereas pre-immune sera did not (data not shown). The specificity of the mouse pAbs was also tested at the molecular level by Western blotting (Fig. 4). Each mouse pAb recognized the macrophage cell membrane protein with which the animals had been immunized. Pre-immune sera did not recognize any of the putative DEN-2 receptors.



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Fig. 4. Specificity of mouse pAbs raised against four putative receptors from DEN-2. Human monocyte-derived macrophage cell membrane proteins were separated by 12% SDS–PAGE and transferred to nitrocellulose. Nitrocellulose strips were incubated in the presence of a mixture of pre-immune sera (lane 1), anti-67K pAb (lane 2), a mixture of anti-67K and anti-87K pAbs (lane 3), a mixture of anti-45K and 67K pAbs (lane 4), and a mixture of anti 27K, anti-45K, anti-67K and anti-87K pAbs (lane 5), followed by goat anti-mouse IgG–PO. Nitrocellulose sheets were treated with ECL reagent and autoradiographed. Western blots are representative of three independent experiments.

 
After confirming the specificity of each pAb, the pAbs were used in two different DEN-2 infection inhibition assays: flow cytometry and immunofluorescence and optical scanning, as described in Methods. Fig. 5 shows a representative (out of three experiments) flow cytometry histogram of NS1 intracellular staining (indicative of DEN-2 replication). A shift to a lower fluorescence intensity in the cell cultures previously exposed to the pAbs as compared to that of untreated cells was also observed, indicating inhibition of DEN-2 replication.



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Fig. 5. Inhibition of DEN-2 infection by mouse polyclonal immune sera recognizing each of the four putative DEN-2 receptors. Human monocyte-derived macrophages were incubated separately in the presence of each of the four mouse pAbs (anti-27K, anti-45K, anti 67K and anti-87K), pre-immune sera or in medium alone for 1 h before being infected with DEN-2. Infection was allowed to proceed for 72 h and virus replication was assessed (by flow-cytometry) by measuring the intracellular expression of DEN-2 NS1: the anti-NS1 mAb 1A12.3 followed by goat anti-mouse IgG–FITC antibodies were used. (A) Flow cytometry histograms of NS1 intracellular expression; (B) percentage of infected cells (NS1-positive cells) and inhibition of infection (in brackets) for each condition. Results are representative of three independent experiments.

 
Flow cytometry analysis showed that DEN-2 infected approximately 84% of untreated human macrophages (positive control), whereas pre-treatment with the anti-27K pAb caused a drop in infection to 21% (75% inhibition), anti-45K pAb to 26% (69% inhibition), anti-67K pAb to about 35% (59% inhibition) and anti-87K pAb to about 56% (44% inhibition). Flow cytometry analysis of DEN-2 infection of undifferentiated and PMA-differentiated U-937 cells was also performed. On average, 1% of undifferentiated and 6% of PMA-differentiated U-937 cells were infected in the absence of pAbs (data not shown). Accordingly, only human macrophages were used for the antibody-mediated infection inhibition assays.

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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Fig. 6. pAb-mediated infection inhibition of human macrophages by DEN-2. Monocyte-derived macrophages were cultured in Lab-Tek chamber glass slides and exposed to each of the four pAbs raised against the putative receptors for DEN-2 or to the corresponding pre-immune sera before infection with DEN-2. After 72 h, cells were permeabilized and labelled with the anti-E protein mAb 2C5.1 and goat anti-mouse IgG–FITC. (A) Fluorescence intensity in each culture condition (as indicative of infection) was assessed in a Typhoon 8600 optical scanner. Confocal microscope images of DEN-2-infected macrophages (B), pre-immune 67K pAb-treated macrophages plus DEN-2 (C) and anti-67K pAb-treated macrophages plus DEN-2 (D).

 
Therefore, mouse pAbs raised against all four putative receptors for DEN-2 were able to inhibit DEN-2 infection of human macrophages, presumably by blocking virus entry via the putative receptors described in this study.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Considerable progress has been made in identifying both the target organs (Couvelard et al., 1999 ; Kurane et al., 1994 ) and the target cells (Avirutnan et al., 1998 ; Bonner & O’Sullivan, 1998 ; Despres et al., 1998 ; Diamond et al., 2000 ; Ho et al., 2001 ; Marianneau et al., 1997 ; Wu et al., 2000 ) for DEN.

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 virus–antibody 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.


   Acknowledgments
 
M.M.B.M.A. was supported by a Consejo Nacional de Ciencia y Tecnología fellowship and is an SNI fellow. We thank Drs Oscar Rojas, Bulmaro Cisneros and Efrain Garrido for helpful advice and discussion, Professor Michael Steward for the critical reading of the manuscript, Dr Jorge A. Sosa for confocal microscopy, Rosalinda Tovar for technical assistance and the Military Hospital Blood Bank personnel for providing blood samples. F.J.S.G. is a COFAA, EDI and SNI fellow. M.L.M. is an SNI fellow.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Avirutnan, P., Malasit, P., Seliger, B., Bhakdi, S. & Husmann, M. (1998). Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. Journal of Immunology 161, 6338-6346.[Abstract/Free Full Text]

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Received 5 September 2001; accepted 4 January 2002.