Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rodrigo, W. W. S. I. Articles by Schlesinger, J. J. Search for Related Content PubMed PubMed Citation Articles by Rodrigo, W. W. S. I. Articles by Schlesinger, J. J.

 Previous Article  |  Next Article 

Journal of Virology, October 2006, p. 10128-10138, Vol. 80, No. 20
0022-538X/06/$08.00+0     doi:10.1128/JVI.00792-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Differential Enhancement of Dengue Virus Immune Complex Infectivity Mediated by Signaling-Competent and Signaling-Incompetent Human FcRIA (CD64) or FcRIIA (CD32)

W. W. Shanaka I. Rodrigo,¹ Xia Jin,^2,3 Shanley D. Blackley,² Robert C. Rose,^2,3 and Jacob J. Schlesinger^2,3*

Departments of Pathology and Laboratory Medicine,¹ Medicine,² Microbiology and Immunology, University of Rochester, Rochester, New York 14642³

Received 18 April 2006/ Accepted 3 August 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fc receptor (FcR)-mediated entry of infectious dengue virus immune complexes into monocytes/macrophages is hypothesized to be a key event in the pathogenesis of complicated dengue fever. FcRIA (CD64) and FcRIIA (CD32), which predominate on the surface of such dengue virus-permissive cells, were compared for their influence on the infectivity of dengue 2 virus immune complexes formed with human dengue virus antibodies. A signaling immunoreceptor tyrosine-based activation motif (ITAM) incorporated into the accessory -chain subunit that associates with FcRIA and constitutively in FcRIIA is required for phagocytosis mediated by these receptors. To determine whether FcRIA and FcRIIA activation functions are also required for internalization of infectious dengue virus immune complexes, we generated native and signaling-incompetent versions of each receptor by site-directed mutagenesis of ITAM tyrosine residues. Plasmids designed to express these receptors were transfected into COS-7 cells, and dengue virus replication was measured by plaque assay and flow cytometry. We found that both receptors mediated enhanced dengue virus immune complex infectivity but that FcRIIA appeared to do so far more effectively. Abrogation of FcRIA signaling competency, either by expression without -chain or by coexpression with -chain mutants, was associated with significant impairment of phagocytosis and of dengue virus immune complex infectivity. Abrogation of FcRIIA signaling competency was also associated with equally impaired phagocytosis but had no discernible effect on dengue virus immune complex infectivity. These findings point to fundamental differences between FcRIA and FcRIIA with respect to their immune-enhancing capabilities and suggest that different mechanisms of dengue virus immune complex internalization may operate between these FcRs.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The interaction between virus and antibody ordinarily leads to neutralization, but the infectivity of some antibody-coated viruses may be enhanced if susceptible cells bear Fc receptors (FcR). This apparent paradox is of particular interest with respect to the dengue viruses: serious forms of dengue fever, manifested by heightened viremia levels and generalized microvascular leak syndromes (53), have been linked to enhanced infection of monocytes/macrophages by dengue virus immune complexes (10, 19). The nature of enhancing antibodies has been widely investigated using primary monocytes/macrophages or macrophage-like cell lines that express FcR. Receptor properties that might affect immune enhancement, however, have received comparatively much less attention largely because heterogeneous FcR display on such cells complicates the interpretation of experimental results.

FcR comprise a multigene family of integral membrane glycoproteins that exhibit complex activation or inhibitory effects on cell functions after aggregation by complexed immunoglobulin G (IgG) (3, 34, 40, 45). Here, we are concerned with two activatory human FcR of different classes and with distinctive but overlapping distribution among monocytes known to be permissive to dengue virus infection. The first, FcRIA (CD64), is a 72-kDa protein found exclusively on antigen-presenting cells of macrophage and dendritic cell lineages, most of which are permissive to dengue virus replication (6, 23, 57). FcRIA exhibits high affinity for monomeric IgG1 and exists bound to this immunoglobulin in vivo. The second, FcRIIA (CD32), is a 40-kDa protein unique to humans and more broadly distributed among a variety of myelogenous cell types. It has low affinity for monomeric IgG, preferentially binding multivalent IgG (27). Each FcR is composed of three domains: an extracellular domain of two (FcRIIA) or three (FcRIA) IgG-like domains, a short hydrophobic transmembrane region, and a cytoplasmic tail. A conserved immunoreceptor tyrosine-based activation motif (ITAM) links each FcR to tyrosine kinase-activated signaling pathways that modulate cell metabolism and physical behavior when triggered by receptor clustering (5, 25, 49, 50). FcRIA acquires this function by noncovalent association with the -chain subunit, a short (ca. 11-kDa) transmembrane ITAM-containing homodimer (22). FcRIIA, unlike other Fc receptors and most immunoreceptors, incorporates the ITAM into its ligand binding chain.

Signal transduction triggered by ligand engagement is intimately involved in the phagocytosis of IgG-opsonized particles where the molecular details of FcRIA and FcRIIA signaling have been revealed in exquisite detail (8, 17, 18, 25, 49). A signaling requirement for the entry of infectious virus immune complexes following FcR engagement is less certain and has been rarely studied. One view is that FcR may facilitate the entry of dengue virus immune complexes by simply concentrating them onto a putative dengue virus receptor, in essence a passive effect that leads to internalization and infection perhaps uninfluenced by FcR signal transduction (26). Conversely, evidence of differential immune enhancement levels among FcR or for modulation of dengue virus immune complex infectivity by FcR-triggered signaling would have important implications with respect to mechanisms of dengue neutralization and dengue fever pathogenesis.

FcRIA and FcRIIA have previously been shown to facilitate antibody-mediated dengue enhancement in human macrophage-like cells by using surrogate plaque assays to measure virus replication (20, 24) since dengue virus does not form plaques in such cells (38). Here, we have examined the relative efficiency with which each of these receptors individually enhances dengue virus immune complex infectivity and have inquired whether signal transduction competency plays a role. Our strategy for answering these fundamental questions surrounding the immune enhancement phenomenon involved the expression of native and mutant forms of human -chain/FcRIA and FcRIIA in dengue virus-permissive COS cells in which dengue virus immune enhancement was directly measured by conventional plaque assay. We found that the infectivities of dengue virus immune complexes were strikingly greater after the engagement of FcRIIA than after that of FcRIA and that signaling competency was required for optimally enhanced infectivity subserved by FcRIA but apparently not for that subserved by FcRIIA.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and dengue viruses. African green monkey kidney-derived COS-7 (fibroblast) or Vero (epithelial) cells were grown in Dulbecco's modified Eagle's medium (DMEM) or MEM, respectively. THP-1 cells kindly provided by Melanie Wellington (University of Rochester, Rochester, NY) were grown in RPMI in stationary culture. C6/36 Aedes albopictus mosquito cells were grown at 28°C in MEM supplemented with sodium pyruvate and nonessential amino acids. Media were supplemented with fetal bovine serum, and cells were grown in a 5% CO₂ atmosphere. Virulent strain 16681 dengue 2 virus (11) and strain New Guinea C (NGC) dengue 2 virus, attenuated by multiple passage in suckling mouse brain (41), were gifts of Walter Brandt and Tadeusz Kochel (Walter Reed Army Institute of Research, Washington, DC, and U.S. Naval Medical Research Center, Bethesda, MD, respectively). Each virus was propagated in mosquito cells and its titer determined by plaque assay in Vero cells.

Dengue virus antibodies. Convalescent anti-dengue virus sera from Thai or Puerto Rican dengue fever patients, gifts from Eric Henchal (Armed Forces Institute for Medical Research, Bangkok, Thailand) and Gladys Sather (Centers for Disease Control, Puerto Rico), respectively, were pooled using equal amounts from each of six subjects; the pool exhibited broad dengue virus serotype neutralizing and hemagglutination-inhibiting activity. IgG1 mouse monoclonal antibodies (MAbs) (J. J. Schlesinger, unpublished) against dengue 2 virus NS1 (MAb 9A9) or envelope E protein (MAb 7E1) were used to detect dengue virus replication in plaque or flow cytometry assays, respectively. MAb 7E1 was purified by affinity chromatography (Pierce Chemicals) and labeled with Alexa 647 (Molecular Probes, Invitrogen Corp.) according to the manufacturers' instructions.

Construction of signaling-competent and signaling-incompetent -chain/FcRIA or FcRIIA vectors. Human FcRIA (30) and -chain (22) cDNA were generously provided by Clark L. Anderson (Ohio State University, Columbus, OH) and Jean-Pierre Kinet (Harvard University, Cambridge, MA), respectively. FcRIIA (H131 allotype) (49) was provided by Jan G. J. van de Winkel (University Hospital Utrecht). To arrange for coordinated expression of FcRIA with -chain, we used a PCR-based strategy to construct a bicistronic expression cassette in a pcDNA5/FLP recombination target (FRT) backbone that contained the coding sequences for -chain and FcRIA in the upstream and downstream positions, respectively, separated by an internal ribosomal entry site derived from encephalomyocarditis virus and expressed under the control of the cytomegalovirus immediate-early promoter. Control constructs were generated by site-directed mutagenesis using standard methods (QuikChange II; Stratagene, La Jolla, CA) for inserting stop codons within -chain or FcRIA. Similar methods were used to generate constructs that contained single, double, or triple ITAM tyrosine residue mutations in multiple permutations. FcRIIA was also generated in the pcDNA5/FRT backbone in monocistronic form. Sequences for all constructs were verified by DNA sequence analysis.

Transient Fc receptor expression in COS cells. Purified recombinant plasmids were transfected into COS cell monolayers by using standard methods (Lipofectamine 2000; Invitrogen, Carlsbad, CA). Cell cultures were trypsinized 48 h after transfection, washed with phosphate-buffered saline (PBS), and kept on ice for immediate use. FcR expression was verified by rosette assay using sheep red blood cells (SRBC) opsonized with rabbit IgG anti-SRBC. The percentage of cells expressing FcR was assessed by counting SRBC rosettes in a hemacytometer and by flow cytometry.

Flow cytometry. THP-1 cells and COS transfectants were washed with PBS and stained with R-phycoerythrin (PE)-conjugated IgG1 monoclonal antibodies against human FcRIA (CD64 MAb 10.1; eBioscences, San Diego, CA) or FcRIIA (CD32 MAb AT10; Serotec, Raleigh, NC) using an R-PE-labeled IgG1 isotype control from the corresponding manufacturer. Stained cells were fixed with 1% paraformaldehyde and analyzed by FACSCalibur using CellQuest software (BD Immunocytometry Systems, Franklin Lakes, NJ); a minimum of 20,000 events was collected from each sample for analysis. The number of FcRIA or FcRIIA molecules expressed on the surface of COS transfectants and THP-1 cells was determined by a quantitative immunofluorescence method that employed standardized QuantiBRITE-PE beads (BD Pharmingen, San Jose, CA), following the manufacturer's instructions. Briefly, the fluorescent intensity of PE-labeled beads was used to establish a standard curve. The number of cell surface FcRIA and/or FcRIIA molecules per cell was then extrapolated from the standard after subtracting for background staining of the IgG1 isotype control. Dengue virus-infected COS cells were quantified by flow cytometry after direct staining of fixed permeabilized cells with Alexa 647-labeled MAb 7E1 against dengue 2 virus envelope E protein.

Western blotting. COS cell transfectants were lysed in Tris saline buffer containing 1% NP-40 and protease inhibitors. Soluble proteins were reduced in Laemmli buffer and separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for transfer to nitrocellulose membranes and immunoblotting with rabbit IgG anti-human -chain (Upstate Cell Signaling Solutions, Lake Placid, NY) and chemiluminescence-based detection (ECL; Amersham Biosciences, Piscataway, NJ). -Chain abundance was quantified by gel-scanning densitometry using ImageQuant version 5.2 software (Molecular Dynamics).

Measurement of opsonized particle binding and phagocytosis. Sheep red blood cells were sensitized with a subagglutinating dilution of rabbit IgG anti-sheep RBC and incubated with transfected COS cells in suspension for 18 h at room temperature before being counted in a hemacytometer chamber; SRBC rosettes were expressed as the percentage of cells with at least three SRBC bound. Surface binding and phagocytosis of opsonized Candida albicans were distinguished by a previously described quantitative double-fluorescence method (55). Briefly, heat-killed yeast cells were stained with fluorescein isothiocyanate (FITC) and sensitized with rabbit antiserum. To measure phagocytosis, transfected COS cells were incubated with opsonized FITC-labeled yeast particles for 45 min at 4°C followed by low speed centrifugation and incubation for 45 min at 37°C before being counterstained with ethidium bromide. In parallel, mixtures of COS transfectants and yeast particles were centrifuged and incubated at 4°C to determine cell surface binding. Cell surface-bound yeast particles were counterstained yellow by ethidium bromide, but internalized FITC-stained yeast particles continued to fluoresce green since ethidium bromide cannot penetrate viable cells. The phagocytic activity of COS transfectants and THP-1 cells was expressed as the phagocytic index, the number of opsonized yeast particles ingested per 100 FcR-expressing cells (25). Cell preparations were photographed at a magnification of x40 with an Olympus BX41TF fluorescence microscope equipped with a digital camera using Qcapture 2.0 software. Images were prepared in Adobe Photoshop CS.

Measurement of dengue 2 virus replication by plaque assay. Preformed dengue virus immune complexes were prepared by incubating mixtures of serially diluted virus or human pooled dengue virus antibody, in checkerboard fashion, for 75 min at 37°C before mixing with 2 x 10⁵ trypsinized COS transfectants suspended in 24-well polystyrene cluster plates. After overnight incubation at 37°C, cell monolayers were washed with PBS and overlaid with 0.6% agarose (SeaKem GTG; FMC BioProducts, Rockland, ME). Agarose plugs were removed 3 days later, and cells were fixed with a 1:1 (vol/vol) acetone-methanol mixture. Dengue virus plaques developed with anti-dengue 2 virus NS1 MAb and a nickel-horseradish peroxidase-based detection method (Vectastain ABC kit; Vector Laboratories, Burlingame, CA), were counted with the aid of a 10x magnifying glass or by scanning the cluster plate into Adobe Photoshop CS for further magnification. In some assays, the addition of fresh medium to the washed COS transfectant monolayers substituted for the agarose overlay. At various times, supernatants were collected for virus titration in Vero cells and cell monolayers trypsinized for analysis by flow cytometry.

Statistics. Student's t test and analysis of variance were performed using MS Excel and SigmaStat software, v3.0 (SPSS, Inc.), respectively.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Signaling-competent and signaling-incompetent FcRIA and FcRIIA expressed in COS cells. FcRIIA exists in two functionally different allelic forms that are determined by a single His/Arg residue at position 131 (54); we selected the H131 form for the present studies because it, unlike the R131 form, efficiently binds IgG2 in addition to the other human IgG subclasses. The sequence for each gene construct was verified by comparing it with that published in GenBank. The FcRIA and FcRIIA gene constructs used in these experiments are presented in Fig. 1A. Earlier investigations that defined a -chain signaling requirement for FcRIA-mediated phagocytosis by COS transfectants have employed separate vectors or FcRIA--chain chimeras to express these genes (17, 25). To ensure uniform FcRIA and -chain coexpression in transfected cells, we incorporated the respective genes into a bicistronic vector, with -chain being inserted upstream of FcRIA so that FcRIA transfectants detected by rosette formation with opsonized particles or by flow cytometry using anti-CD64 monoclonal antibodies were also sure to contain -chain. This gene arrangement would be predicted to result in expression of -chain in excess of FcRIA (roughly by a factor of 2), presumably due to the inherent inefficiency of ribosomal entry (13, 29). A stoichiometric -chain excess was desired since each transmembrane FcRIA monomer associates with a -chain dimer to form the functional complex. It also served to increase our confidence that cells exhibiting surface expression of FcRIA by flow cytometry would also likely contain -chain which is largely intracellular and therefore cannot be detected by this method without permeabilization. Stop codons were inserted into the FcRIA or ^WT-chain sequence of bicistronic constructs (Fig. 1A) to provide control vectors. The -chain cytoplasmic tail incorporates three tyrosine residues: one (Y58) upstream of the ITAM and two (Y65 and Y76) in the ITAM. The FcR IIA cytoplasmic tail has an analogous tyrosine residue distribution (upstream Y280; ITAM Y287 and Y303). Earlier molecular dissection of phagocytosis indicated that the upstream non-ITAM tyrosine residue also contributed to this function (14, 18, 31). To ensure the abrogation of the signaling competency of the -chain/FcRIA and FcRIIA constructs, we therefore mutated the three potentially activating tyrosine residues of each receptor (^3XMUT/FcRIA and FcRIIA^3XMUT). -Chain expression was assessed by Western blotting (Fig. 1B); equivalence of -chain abundance among the -chain transfectants was confirmed by scanning densitometry. Flow cytometry was used to verify and to measure FcR expression and to determine the number of FcR molecules on the cell surface. THP-1 cells, a human monocyte line that constitutively expresses FcRIA and FcRIIA exclusively (9), were used as a control (Fig. 1C and Table 1). The percentages of FcRIA- and FcRIIA-expressing COS cells were comparable within the panel of transfectants (Table 1) and were at least two- to threefold higher than those previously obtained using a DEAE-dextran transfection method (25, 42). THP-1 cells expressed 5,500 FcRIA and 58,000 FcRIIA surface molecules per cell, amounts that are in agreement with published data (9). The number of FcRIA or FcRIIA molecules expressed on COS cells was not affected by Tyr-to-Phe mutations in the associated -chain or FcRIIA cytoplasmic tails, respectively. The average number of cell surface FcRIIA molecules (43,000) was greater than that of FcRIA molecules associated with -chain (30,000), but this difference was not statistically significant (P > 0.10; two-tailed t test). Remarkably, the number of surface FcRIA molecules was 50% higher (P < 0.03; two-tailed t test) when this receptor was associated with -chain than when it was expressed without it.

View larger version (16K): [in this window] [in a new window]

FIG. 1. Structure and expression of -chain/FcRIA complex and FcRIIA versions in COS transfectants. (A) The order of -chain and FcRIA genes in the bicistronic construct ensured that FcRIA-expressing COS cells also expressed the -chain (WT/FcRIA). An encephalomyocarditis virus-derived internal ribosomal entry site (IRES) drives the internal initiation of the FcRIA gene. Other genes are expressed under the control of a cytomegalovirus immediate-early promoter. Stop codons inserted into the FcRIA or WT-chain sequence of bicistronic constructs provided control vectors. FcRIIA was cloned into the same pcDNA5/FRT to generate a monocistronic construct (FcRIIAWT and FcRIIA3XMUT). A consensus Kozak sequence was introduced upstream of the -chain and FcRIIA genes. Tyrosine residue positions in ITAMs of -chain and FcRIIA are numbered, starting from the +1 start. (B) Solubilized lysates prepared from 2.5 x 105 cells of each COS transfectant were electrophoresed and subjected to Western blotting using a monospecific rabbit serum against human -chain (22). (C) PE-labeled CD32 (MAb AT10) or CD64 (MAb 10.1) monoclonal antibodies and PE-labeled mouse IgG1 were used to measure the proportions of COS transfectants expressing the respective FcR. The THP-1 human macrophage cell line served as a control. Results are representative of five or six determinations for FcRIA transfectants and three determinations for FcRIIA transfectants (Table 1).

View this table: [in this window] [in a new window]

TABLE 1. Expression of human FcRIA (CD64) and FcRIIA (CD32) in COS-7 cells analyzed by flow cytometry

FcR surface expression and signaling competency verified by binding and phagocytosis of opsonized yeast particles. Signaling-incompetent FcRIA or FcRIIA transfectants bind opsonized SRBC but are not efficiently phagocytic (17, 25, 48). If properly constructed, our ^WT/FcRIA and FcRIIA^WT COS transfectants were expected to perform both functions, whereas transfectants appropriately mutated or expressing FcRIA without -chain were expected to only bind IgG-coated particles. We measured binding and phagocytosis of IgG-opsonized yeast particles by COS cells that expressed FcRIA or FcRIIA to verify receptor functional activity and used phagocytic THP-1 cells as a control. Fluorescence microscopy has been validated as an accurate method for estimating phagocytosis (25). To simultaneously measure FcR-mediated binding and phagocytosis, we adopted a quantitative double- fluorescence method that employed IgG-opsonized, FITC-stained Candida albicans particles and ethidium bromide counterstaining to distinguish between THP-1 cell surface-bound and internalized particles (55). Figure 2A illustrates the appearance of surface-bound (yellow) or internalized (green) fluorescent-stained yeast particles after incubation with THP-1 cells or COS cells that expressed /FcRIA or FcRIIA. COS cells transfected with the control empty vector or ^WT/FcRIA^STP did not bind opsonized particles. In accord with receptor expression measured by flow cytometry (Table 1), more THP-1 cells (90%) bound opsonized yeast particles than did the FcR transfectants, where the levels were similar (50 to 60%). Comparable results were obtained with opsonized SRBC (data not shown). COS cells expressing FcR versions that were predicted to be signaling-competent (^WT/FcRIA or FcRIIA^WT) exhibited significantly greater phagocytic capacity than did cells expressing the respective signaling-incompetent versions (^3XMUT/FcRIA or FcRIIA^3XMUT) (Fig. 2B).

View larger version (27K): [in this window] [in a new window]

FIG. 2. Binding and phagocytosis of opsonized C. albicans by COS cells expressing FcRIA or FcRIIA. Rabbit IgG-sensitized, fluorescein isothiocyanate-stained yeast particles were incubated with COS cells expressing signal-competent (WT/FcRIA and FcRIIAWT) or signal-incompetent (3XMUT/FcRIA, STP/FcRIA, and FcRIIA3XMUT) FcR. COS cells expressing -chain only or transfected with the pcDNA5/FRT vector served as controls. Phagocytosis by human macrophage-like THP-1 cells that express both FcR was measured in parallel in each experiment. Binding and phagocytosis of opsonized C. albicans were measured using a quantitative double-fluorescence technique that employed ethidium bromide to selectively stain cell-bound (yellow) but not internalized (green) fluorescein isothiocyanate-stained yeast particles (see Materials and Methods). (A) Immunofluorescent photomicrographs (x40) of FcR and control cells incubated with opsonized yeast particles. (B) Phagocytosis is expressed as the phagocytic index, the number of internalized yeast particles per 100 FcR-expressing COS cells. P values: a, P < 0.02 (THP-1 versus WT/FcRIA or FcRIIAWT); b, P < 0.03 (3XMUT/FcRIA versus WT/FcRIA); c, P < 0.003 (FcRIIA3XMUT versus FcRIIAWT). Results are the means and standard deviations for three individual experiments with FcRIA and four individual experiments with FcRIIA, performed in duplicate.

Collectively, our results indicated that the FcR and -chain genes of interest were properly constructed, correctly expressed, and functional with respect to binding and internalization of IgG-opsonized particles. Having confirmed that these properties of a professional macrophage were conferred on COS cells, we next investigated their interaction with dengue virus immune complexes.

Dengue virus immune complex infectivity is greater in COS cells expressing FcRIIA than in those expressing FcRIA. Pooled human anti-dengue virus convalescent-phase sera with broad dengue virus serotype-neutralizing and hemagglutination-inhibiting capacity were used to prepare infectious dengue 2 virus immune complexes for presentation to the respective COS FcR transfectants. This polyclonal serum pool, prepared from serologically screened American and Asian dengue fever patients, likely represents broad dengue virion antigenic specificity and IgG subclass diversity (47), so that any differences in results among COS transfectants should confidently reflect behavior specific to the respective FcR. We used two strains of dengue 2 virus to prepare immune complexes: (i) a virulent strain, 16681, isolated from a patient with dengue hemorrhagic fever/shock syndrome during a South Asian epidemic that was marked by a high prevalence of complicated dengue fever (11) and (ii) the prototypic attenuated strain, New Guinea C (NGC) (41). We measured the infectivities of preformed dengue 2 virus immune complexes in FcRIA- or FcRIIA-expressing COS cells by a conventional flavivirus plaque reduction neutralization assay method performed by infecting cells in suspension (32). Cells were thus continuously exposed to virus immune complexes during the initial monolayer formation. Strain 16681 dengue 2 virus produced small (<1 mm), relatively homogeneous, and sharply defined plaques in COS cells, whereas those formed by the NGC strain were larger (2 mm) and more irregular. The efficiencies of dengue 2 virus plaque formation, in the absence of antibodies, were comparable among the FcR and control (empty vector, ^WT/FcRIA^STP) transfectants (Fig. 3). Figure 4 shows the relative infectivities of strain 16681 (panel A) and NGC (panel B) dengue 2 virus immune complexes, respectively, in signaling-competent (^WT/FcRIA and FcRIIA^WT) or signaling-incompetent (^3XMUT/FcRIA and FcRIIA^3XMUT) COS transfectants. COS cells transfected with the pcDNA5/FRT "empty" vector or with -chain only (^WT/FcRIA^STP) served as controls. In 10 such experiments performed in duplicate or triplicate, the infectivities of partially neutralized dengue virus immune complexes were enhanced in both /FcRIA- and FcRIIA-expressing COS cells, but this effect was consistently and strikingly greater in FcRIIA than in FcRIA transfectants. The abrogation of FcRIA signaling competency by mutation of all -chain cytoplasmic-tail Tyr residues led to reduced dengue virus immune complex infectivity, but the mutation of the analogous FcRIIA cytoplasmic tyrosine residues had no apparent effect. We observed no difference between the two dengue virus strains with respect to the degrees of enhanced immune complex infectivity among the FcR transfectants. To further compare the relative importance of signal transduction capacity for FcRIA- and FcRIIA-mediated enhancement, we presented immune complexes formed with a range of antibody and dengue 2 virus concentrations to COS cells expressing the respective native and mutant receptors (Fig. 4C and D). We used strain 16681 dengue 2 virus for these experiments because its distinctive plaque morphology allowed for more-precise counting than did strain NGC, and we adjusted the antibody and virus concentrations such that the numbers of plaques produced were in a range that permitted comparative counting among the respective COS transfectants in the same assay. Enhanced immune complex infectivity was observed only in FcR-expressing cells (independent of receptor signaling competency) compared to what was found for control transfectants. Dengue virus immune complex infectivity was significantly greater in FcRIIA- than in FcRIA-expressing COS cells over the range of virus and antibody concentrations examined. Complete abrogation of signaling competency significantly diminished FcRIA-enhanced infection but, remarkably, had no discernible effect on immune complex infectivity enhanced by FcRIIA engagement.

View larger version (79K): [in this window] [in a new window]

FIG. 3. Efficiencies of plaquing are comparable among COS transfectants. COS transfectants were infected with serial twofold (25 to 200) PFU dengue 2 virus (16681) in the absence of antibody. (A) Plaques were detected by indirect immunostaining with a dengue 2 virus NS1-specific monoclonal antibody. (B) The efficiencies of virus plaque formation were comparable (P = 0.17) among FcRIA, FcRIIA, and control (empty vector, WT/FcRIASTP) COS transfectants at each virus MOI. There were too many plaques to count at the 200-PFU input. Results are representative of three experiments performed in quadruplicate.

View larger version (76K): [in this window] [in a new window]

FIG. 4. Infectivity of the virulent strain 16681 or attenuated strain New Guinea C dengue 2 virus immune complex is enhanced in COS cells that express FcRIA or FcRIIA. Virus-antibody complexes, prepared with serially diluted human dengue virus antiserum and dengue 2 virus (16681 [A] and NGC [B]), were added to signaling-competent (WT/FcRIA and FcRIIAWT) or signaling-incompetent (3xMUT/FcRIA; FcRIIA3xMUT) Fc receptor-expressing COS cells. COS cells expressing -chain only (WT/FcRIASTP) or cells transfected with the empty pcDNA5/FRT vector served as negative controls. Plaques were detected by indirect immunostaining with a dengue 2 virus NS1-specific monoclonal antibody. Results are representative of 10 individual experiments performed in duplicate or triplicate. (C) Dengue 2 virus (16681) immune complexes were prepared by incubating virus at a single MOI (0.025) with serially diluted human dengue virus antiserum. (D) Immune complexes prepared with a single antibody dilution (1/1,000) and serial virus MOIs were added to signaling-competent (WT/FcRIA and FcRIIAWT) or signaling-incompetent (3XMUT/FcRIA and FcRIIA3XMUT) Fc receptor-expressing COS cells. Cells expressing -chain only (WT/FcRIASTP) or those transfected with the pcDNA5/FRT vector served as negative controls. Plaques were detected by indirect immunostaining with a dengue 2 virus NS1-specific monoclonal antibody. For FcRIIA, plaques corresponding to an MOI of 0.5 were too numerous to count. P values were determined using a two-tailed t test. a, WT/FcRIA versus 3XMUT/FcRIA; b, 3XMUT/FcRIA versus WT/FcRIASTP and vector controls. For panel C: a, P < 0.001; b, P < 0.03. For panel D: a, P < 0.005; b, P < 0.007. Results are the means and standard deviations for an experiment performed in quadruplicate and are representative of three individual experiments performed in triplicate or quadruplicate.

The modulating effect of a signaling-competent -chain on FcRIA-mediated infection was consistent and significant but relatively small (2-fold) in this series of experiments. Therefore, to further verify the effect, we performed three additional experiments. First, we used flow cytometry as an independent method for comparing dengue virus immune complex infectivities between COS cells that expressed FcRIA associated with a signaling-competent or a signaling-incompetent -chain (Fig. 5). COS transfectants were mixed with preformed dengue virus immune complexes or were mock infected in suspension. After overnight incubation, cell monolayers were washed to remove residual virus, and the medium was replenished. Two days later (5 days following transfection), cell monolayers were trypsinized and cells were stained with fluorescence-labeled MAbs against either FcRIA (CD64) or dengue virion envelope E glycoprotein. The proportions of ^WT/FcRIA- and ^3XMUT/FcRIA-expressing cells were essentially equivalent (ca. 25%) but substantially lower than when infection was initiated, 2 days after transfection (ca. 80%) (Table 1), reflecting expression decay and the possibly preferential growth of the nonexpressing cell population. The proportions of infected cells detected among non-FcRIA-expressing cells in the three COS transfectants were also roughly equivalent (1.52% to 1.85%). In contrast, the proportion of dengue virus infected cells expressing ^WT/FcRIA was approximately twofold greater than that of cells expressing ^3XMUT/FcRIA. This observation was highly consistent with results obtained by direct plaque assay.

View larger version (61K): [in this window] [in a new window]

FIG. 5. Infection of signaling-competent and -incompetent, FcRIA-expressing COS cells measured by flow cytometry. COS cells expressing WT/FcRIA or 3XMUT/FcRIA and control vector COS transfectants were infected with preformed dengue virus immune complexes (MOI, 0.25; dengue virus antiserum dilution, 1/4,000) or mock infected and analyzed by flow cytometry 2 days postinfection with Alexa 647-labeled anti-dengue 2 virus envelope E protein (7E1) or PE-labeled anti-CD64 monoclonal antibodies, respectively.

Second, we compared the relative infectivities of dengue virus immune complexes by direct plaque assay in COS cells expressing FcRIA without -chain or FcRIA associated with signaling-competent or signaling-incompetent -chain (Fig. 6A); a virus concentration (multiplicity of infection [MOI], 0.25) and antibody dilution range (1/500 to 1/4,000) that resulted in plaque amounts that approximated those observed with FcRIIA were chosen (Fig. 4C and D). The infectivities of dengue virus immune complexes were up to 20-fold greater in signaling-competent, FcRIA-expressing COS cells than in control COS transfectants. Immune complex infectivity was also significantly enhanced in signaling-incompetent COS cells that expressed FcRIA without -chain or with a mutated -chain (P < 0.001). FcRIA-mediated enhancement was greater when associated with signaling-incompetent -chain than without a -chain, but the difference was not statistically significant (P = 0.06).

View larger version (13K): [in this window] [in a new window]

FIG. 6. FcRIA bereft of -chain mediates dengue virus immune enhancement. Dengue 2 virus (16681) immune complexes prepared by incubating virus at a single MOI (0.25) and human dengue virus antiserum dilution (1/500 to 1/4,000 [A] and 1/4,000 [B]) were incubated in cluster plates overnight with trypsinized COS cells expressing FcRIA alone (STP/FcRIA) or in association with native (WT/FcRIA) or signaling-incompetent -chain (3XMUT/FcRIA). After overnight incubation, an agarose overlay was added to the monolayered cells (A) or monolayers were washed to remove residual virus and replenished with fresh medium (B). In the experiment whose results are shown in panel A, agarose plugs were removed on the second postinfection day and plaques were detected by indirect immunostaining with a dengue 2 virus NS1-specific monoclonal antibody. Each condition was tested in quadruplicate. a, P = 0.06 (3XMUT/FcRIA versus STP/FcRIA); b, P < 0.001 (STP/FcRIA versus vector control). In the experiment whose results are shown in panel B, supernatants were removed in their entirety from the respective wells daily and dengue virus was titrated by plaque assay in Vero cells. Virus concentrations are expressed as the log10 means ± standard deviations for an experiment performed in triplicate and titrated in duplicate. P < 0.003 (vector control versus WT/FcRIA, 3XMUT/FcRIA, and STP/FcRIA); P < 0.015 (WT/FcRIA versus 3XMUT/FcRIA or STP/FcRIA).

Third, we performed an experiment that compared virus replication in FcRIA-expressing and control cells at multiple time points after immune complex inoculation by using a surrogate amplification plaque assay with Vero cells (Fig. 6B). Here, the respective COS transfectants were infected in triplicate with preformed dengue virus immune complexes in 12-well cluster plates (a single plate for each assay day) by using the same protocol as that for the direct plaque assay but omitting the agarose overlay. After overnight incubation, cell monolayers were washed to remove residual virus and were replenished with fresh medium before the first supernatant was collected (day 0) for virus titration. Supernatants were collected in their entirety daily thereafter and stored at –80°C for subsequent titration, in duplicate, in Vero cells. Figure 6B shows an experiment that measured virus replication over the course of 6 days. The results were in accord with those obtained by direct plaque assay with FcRIA-expressing or control cells, with the highest levels of immune complex infectivity observed in cells expressing signaling-competent ^WT/FcRIA. Immune complex infectivity was up to 40-fold greater with the ^WT/FcRIA transfectant than with vector control cells. Significantly enhanced immune complex infectivity was also observed with FcRIA alone or in association with mutated -chain, but at a lower (up to 15-fold) level. There was no significant difference between ^STP/FcRIA and ^3XMUT/FcRIA. These differential effects were observed up to 4 days after infection. Collectively, the results of these experiments indicated a hierarchy of immune complex infectivity in COS FcRIA transfectants, in the following order: ^WT/FcRIA > ^3XMUT/FcRIA ^STP/FcRIA > vector control.

FcRIA-mediated phagocytosis and immune complex infectivity are proportionately reduced by selective -chain mutation. Previous molecular dissection of phagocytosis by COS cells that expressed FcRIA--chain chimeras revealed a hierarchy of effects of -chain Tyr-to-Phe residue changes on phagocytosis (18). To test the hypothesis that FcRIA-mediated phagocytosis and dengue virus immune complex infectivity involve a common mechanism, we prepared a panel of bicistronic vectors consisting of FcRIA and -chain versions with selected tyrosine residue mutations and measured phagocytosis and dengue virus immune complex infectivity in parallel within this COS transfectant panel. COS cells transfected with the empty vector or those expressing only -chain (^WT/FcRIA^STP) or FcRIA (^STP/FcRIA) served as controls. Equivalent FcRIA expression levels among the FcRIA transfectants were verified by flow cytometry; in accord, the -chain abundances measured by Western blotting and densitometry were comparable among the respective COS transfectants (Fig. 7A). We observed equivalent bindings of opsonized yeast particles among the COS cells that expressed FcRIA (data not shown). The quantitative phagocytoses of opsonized yeast particles and relative infectivities of dengue virus immune complexes among the COS transfectants are shown in Fig. 7B. COS transfectants that did not express FcRIA exhibited no phagocytic activity. The highest phagocytic indices were observed in COS cells that expressed FcRIA associated with -chain in native form. COS cells that expressed FcRIA unassociated with -chain had the lowest receptor surface densities and exhibited only trivial phagocytosis. Single or double -chain ITAM tyrosine mutations were accompanied by up to 10-fold reduction in phagocytic activity. Mutation of the -chain non-ITAM tyrosine residue (Y58F) also led to a modest reduction in phagocytic activity. Dengue virus immune complex infectivity was essentially neutralized in control COS cells that did not express FcRIA. Immune complex infectivity was increased more than 10-fold in COS cells that expressed FcRIA associated with -chain in native form. Infectivity was also significantly (P < 0.05; two-tailed t test) increased in COS cells that expressed FcRIA without a -chain, but at a much lower level. All -chain ITAM mutations and the -chain deletion (^STP/FcRIA) led to a modest (30 to 50%), but significant (P < 0.05), reduction in immune complex infectivity compared to that observed with the ^WT/FcRIA transfectant. Point mutation of upstream Tyr residue 58 was associated with reduced immune complex infectivity-enhancing activity that was not significant (P > 0.05) and that did not add to the effect of the ITAM mutations. These comparative findings were consistent over a range of virus and antibody concentrations (data not shown). Both single and double Tyr-to-Phe mutations of the -chain tail ITAM were accompanied by a parallel reduction in phagocytosis and immune complex infectivity. To discern whether phagocytosis and immune complex internalization might have similar mechanisms, we performed a linear regression analysis and found a highly significant correlation (P < 0.01) between phagocytic and immune enhancement capacities within the COS transfectant panel incubated with dengue virus immune complexes formed with serial MOIs of dengue virus (0.25, 0.5, and 1.0) and dengue virus antiserum (1/1,000) (Fig. 7C). Collectively, these data point to a shared pathway for phagocytosis and enhanced dengue virus immune complex infectivity mediated by signaling-competent FcRIA and a second, somewhat less efficient entry mechanism that operates simply by FcRIA engagement.

View larger version (20K): [in this window] [in a new window]

FIG. 7. FcRIA-mediated phagocytosis and dengue virus immune complex infectivity are proportionately reduced by selective -chain mutation. COS cells were transfected with bicistronic vectors composed of -chain alone (WT/FcRIASTP), FcRIA alone (STP/FcRIA), or FcRIA and -chain, in which its cytoplasmic tail residues (Y58, Y65, and Y76) were individually or multiply (Y65,76 and 3XMUT) mutated by Tyr-to-Phe residue substitution. Results are from an experiment performed in triplicate. (A) -Chain abundance determined by Western blotting. Solubilized lysates prepared from 2.5 x 105 cells of each COS transfectant were electrophoresed and subjected to Western blotting using a monospecific rabbit serum against human -chain (22). (B) Phagocytoses of opsonized yeast particles and infectivities of dengue virus immune complexes. COS cells were transfected with -chain/FcRIA versions or FcRIA only. COS cells transfected with an empty vector or expressing -chain only (WT/FcRIASTP) served as controls. The phagocytic index was defined as the number of yeast particles internalized by 100 FcRIA-expressing COS cells. In parallel, the respective COS transfectants were incubated with dengue 2 virus (16681) immune complexes formed with pooled human dengue virus antiserum (1/1,000) and serial concentrations of dengue virus; results for an experiment performed in triplicate with a virus MOI of 1.0 are shown. For both assays, the results were normalized against those for the WT/FcRIA transfectant. All -chain ITAM mutations and the -chain deletion (STP/FcRIA) led to significant (P < 0.05, two-tailed t test) reductions in immune complex infectivity compared to that observed with the WT/FcRIA transfectant. (C) Correlation between phagocytosis and infectivity among dengue virus immune complexes formed with dengue virus antisera (1/1,000 dilution) and dengue 2 virus with MOIs of 0.25 (a), 0.50 (b), and 1.0 (c).

Top Abstract Introduction Materials and Methods Results Discussion References

 
FcR-mediated phagocytosis, internalization of relatively large opsonized particles, and endocytosis, internalization of soluble immune complexes, are biologically distinguishable processes. For both, ligand-clustered receptors are internalized, but only for phagocytosis does FcR signaling competency appear essential for completion of the entry process. For example, cells expressing FcRIA bereft of a -chain or FcRIIA with tail ITAM alterations may internalize soluble IgG complexes but not opsonized particles (4, 25, 35, 48). Virus immune complexes are interesting ligands in this respect, since their sizes and infectivities depend on the nature and quantity of coating antibody (2, 37) and they may have access to other routes of internalization that utilize virus receptors which themselves may trigger signaling events (44).

In this study, we compared the influence of FcRIA and FcRIIA on the infectivities of dengue virus immune complexes prepared with human neutralizing dengue virus antibodies. Signaling-competent and signaling-incompetent versions of these receptors were expressed in dengue virus-permissive COS cells to discern whether dengue virus immune complex internalization, like that of opsonized large particles, depended on the receptors' activation properties.

Our approach using COS transfectants to measure dengue virus immune complex infectivity after Fc receptor engagement offered a number of advantages over studies that have employed macrophages or macrophage-like cell lines. First, FcRIA and FcRIIA were examined individually in isolation from other FcR classes or unrelated macrophage receptors that may alter their function on such cells (3, 33, 36). Second, FcRIA and FcRIIA concentrations on the surfaces of COS transfectants were comparable (30,000 to 40,000 molecules per cell), which is generally not the case for monocytes/macrophages in which the abundance of these receptors is differentially regulated by inflammatory mediators and affected by culture conditions (15, 51), e.g., the surface concentrations of FcRIIA on unstimulated THP-1 cells were 10-fold higher than those of FcRIA (Table 1). Our determinations of FcRIA and of FcRIIA COS cell surface concentrations were within the range reported for FcR on human peripheral blood monocytes/macrophages (15, 51). Interestingly, we found that FcRIA surface concentrations were significantly higher when this receptor was associated with -chain than without it, in accord with the -chain requirement for efficient FcRIA assembly and surface expression in vivo (45, 46, 52) and in contrast to the reduced FcRIA expression in COS cells when separate vectors were used to deliver FcRIA and -chain genes (30). That the pattern of FcRIA expression in our transfectants appeared to more closely reflect the in vivo condition suggests that a precise stoichiometry between -chain and FcRIA may be required for faithful receptor display and that our bicistronic vector design satisfied this requirement. Since FcRIA and -chain are noncovalently linked at the transmembrane level (12, 17, 30), it is unlikely that mutations in the -chain cytoplasmic domain affected this association; that the FcRIA surface concentration was the same when associated with a native or mutated -chain lent further support to this conclusion. The abrogation of Fc receptor signaling competency was verified by the significant reduction in phagocytic activity upon ITAM mutation. Serial Tyr-to-Phe mutations in the -chain cytoplasmic tail resulted in reduced phagocytic activities for yeast particles by FcRIA/-chain complex-expressing COS cells, similar to those for SRBC by COS cells expressing the receptor complex in the form of a chimeric protein (18). Low-level yeast internalization observed among COS fibroblasts expressing signaling-incompetent FcR transfectants was not too surprising, since many fibroblast cell types display "nonprofessional" phagocytic properties without Fc receptors (39) and COS cells that expressed FcRIA without -chain have exhibited similar levels of opsonized SRBC internalization (25). It is also quite possible that yeast particles may be inherently more susceptible to internalization than SRBC when concentrated onto the fibroblast cell surface by FcR engagement.

The extracellular portion of FcRIA was earlier reported to be sufficient for increased dengue virus immune complex infectivity in COS cells, but a concurrent -chain modulating effect was not observed (42). This result was postulated due to reduced receptor density that attended cotransfection with -chain and FcRIA in separate vectors. In the present experiments, we have revisited the question of a possible -chain role in dengue virus immune enhancement by using a significantly more efficient transfection method that resulted in two- to threefold-higher expression levels and bicistronic vectors designed to ensure uniform coexpression of FcRIA and -chain versions among the cotransfectants which we have confirmed biochemically and by flow cytometry. This new methodology provided robust assays capable of more-precise discrimination of the relatively small differences (<3-fold) in dengue virus immune complex infectivity observed among the FcRIA/-chain constructs compared to the much larger differences (>10-fold) that we observed between FcRIA and FcRIIA. Using three complementary methods to determine dengue virus immune complex infectivity in FcRIA-expressing COS cells—direct plaque assay, surrogate virus replication assay, and flow cytometry—we found that enhanced immune complex infectivity mediated by FcRIA was greatest when the receptor was associated with a -chain in its native form and that abrogation of -chain ITAM signaling capacity by Tyr-to-Phe mutation reduced but did not entirely eliminate this function. We also confirmed that FcRIA bereft of a -chain is sufficient for enhancement. We interpret our results with FcRIA as reflecting at least two virus immune complex internalization mechanisms at work. The first is a -chain signaling-dependent event wherein infectious virus immune complex aggregates of sufficient size triggered a classical phagocytosis entry pathway. This mechanism is suggested by the correlation between phagocytic capacity and immune complex infectivity among COS cells that expressed FcRIA associated with -chain ITAM mutants. Indeed, antibody-virus complexes, including opsonized flaviviruses, can form lattice structures of considerable size (2, 7), so that for dengue virus (50-nm diameter), immune complexes composed of as few as 10 virions, i.e., a 500-nm "particle," might be predicted to trigger phagocytosis (1). The second is a somewhat less efficient entry mechanism that did not require -chain activation (or, indeed, its presence) and relied simply on concentrating partially neutralized virions onto the cell for entry by a parallel endocytosis mechanism. Importantly, we observed no effect of isolated -chain expression on virus or virus immune complex infectivity, arguing against the enhanced replication explained by -chain association with a cell protein other than FcRIA.

FcRIIA was strikingly more efficient than FcRIA in enhancing dengue virus immune complex infectivity. Abolishing FcRIIA ITAM signaling competency led to impaired phagocytosis, but unlike with signaling-incompetent FcRIA, immune enhancement appeared to be unaffected. Our experiments do not offer an immediate explanation for the divergent findings with these FcR. FcRIA and FcRIIA exhibit differences in relative affinity for IgG subclasses, but it seems somewhat unlikely that skewed anti-dengue 2 virus isotype distribution accounted for the difference in Fc receptor behavior since dengue virus-specific IgG1 and IgG3, which have been found represented in the greatest abundances among IgG subclasses in dengue virus convalescent-phase sera (47), are bound by both receptor types with similar or identical affinities (45, 56). FcRIIA preferentially binds immune complexes and exhibits a high dissociation rate constant (27), whereas FcRIA preferentially binds monomeric IgG with notably high affinity. Ligand-clustered Fc receptors, including FcRIIA, are known to concentrate in cell membrane regions, e.g., lipid rafts, rich in a variety of signaling molecules and potential virus receptor engagement sites (16, 21, 28, 43). It seems reasonable to speculate that FcRIIA is better equipped than is FcRIA to utilize alternative signaling pathways and entry mechanisms made available by relocation to such sites where weakly bound immune complexes might be more easily transferred to favorable entry pathways. Bispecific monoclonal antibodies that directed dengue virus to FcRIIA or non-Fc receptor proteins on the surfaces of U937 human macrophage-like cells enhanced infection, arguably by such an alternate entry mechanism (26).

Our findings emphasize the conditional nature of virus neutralization or enhancement by antibody and suggest an approach for further investigation of an aspect of dengue virus-antibody interaction that is tied to both the protective and the pathological immune response to infection by this virus. Further studies are in progress to explain the apparent divergence in immune enhancement between FcR and to discover to what extent epitope specificity and affinity in the IgG antibodies that comprise the dengue virus immune complex play a role in the outcome of Fc receptor engagement.

 
We thank Lihua Rong and Huiyuan Chen for expert technical assistance.

This work was supported by grants from the Pediatric Dengue Vaccine Initiative (TR03/04 to J.J.S. and TR16 to X.J.).

 
* Corresponding author. Mailing address: Division of Infectious Diseases, Department of Medicine, University of Rochester School of Medicine and Dentistry, Box 689, 601 Crittenden Ave., Rochester, NY 14642. Phone: (585) 275-5871. Fax: (585) 442-9328. E-mail: jacob_schlesinger{at}urmc.rochester.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Aderem, A., and D. M. Underhill. 1999. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17:593-623.[CrossRef][Medline]
2
2. Almeida, J. D., and A. P. Waterson. 1969. The morphology of virus-antibody interaction. Adv. Virus Res. 15:307-338.[Medline]
3
3. Daeron, M. 1997. Fc receptor biology. Annu. Rev. Immunol. 15:203-234.[CrossRef][Medline]
4
4. Davis, W., P. T. Harrison, M. J. Hutchinson, and J. M. Allen. 1995. Two distinct regions of FC gamma RI initiate separate signalling pathways involved in endocytosis and phagocytosis. EMBO J. 14:432-441.[Medline]
5
5. Duchemin, A. M., L. K. Ernst, and C. L. Anderson. 1994. Clustering of the high affinity Fc receptor for immunoglobulin G (Fc gamma RI) results in phosphorylation of its associated gamma-chain. J. Biol. Chem. 269:12111-12117.[Abstract/Free Full Text]
6
6. Fanger, N. A., K. Wardwell, L. Shen, T. F. Tedder, and P. M. Guyre. 1996. Type I (CD64) and type II (CD32) Fc gamma receptor-mediated phagocytosis by human blood dendritic cells. J. Immunol. 157:541-548.[Abstract]
7
7. Fauvel, M., H. Artsob, and L. Spence. 1977. Immune electron microscopy of arboviruses. Am. J. Trop. Med. Hyg. 26:798-807.[Abstract/Free Full Text]
8
8. Fitzer-Attas, C. J., M. Lowry, M. T. Crowley, A. J. Finn, F. Meng, A. L. DeFranco, and C. A. Lowell. 2000. Fcgamma receptor-mediated phagocytosis in macrophages lacking the Src family tyrosine kinases Hck, Fgr, and Lyn. J. Exp. Med. 191:669-682.[Abstract/Free Full Text]
9
9. Fleit, H. B., and C. D. Kobasiuk. 1991. The human monocyte-like cell line THP-1 expresses Fc gamma RI and Fc gamma RII. J. Leukoc. Biol. 49:556-565.[Abstract]
10
10. Halstead, S. B. 1989. Antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenetic cascade. Rev. Infect. Dis. 11(Suppl. 4):S830-S839.[Medline]
11
11. Halstead, S. B., and P. Simasthien. 1970. Observations related to the pathogenesis of dengue hemorrhagic fever. II. Antigenic and biologic properties of dengue viruses and their association with disease response in the host. Yale J. Biol. Med. 42:276-292.[Medline]
12
12. Harrison, P. T., L. Bjorkhaug, M. J. Hutchinson, and J. M. Allen. 1995. The interaction between human Fc gamma RI and the gamma-chain is mediated solely via the 21 amino acid transmembrane domain of Fc gamma RI. Mol. Membr. Biol. 12:309-312.[Medline]
13
13. Hennecke, M., M. Kwissa, K. Metzger, A. Oumard, A. Kroger, R. Schirmbeck, J. Reimann, and H. Hauser. 2001. Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs. Nucleic Acids Res. 29:3327-3334.[Abstract/Free Full Text]
14
14. Indik, Z. K., J. G. Park, S. Hunter, and A. D. Schreiber. 1995. The molecular dissection of Fc gamma receptor mediated phagocytosis. Blood 86:4389-4399.[Abstract/Free Full Text]
15
15. Jungi, T. W., and S. Hafner. 1986. Quantitative assessment of Fc receptor expression and function during in vitro differentiation of human monocytes to macrophages. Immunology 58:131-137.[Medline]
16
16. Katsumata, O., M. Hara-Yokoyama, C. Sautes-Fridman, Y. Nagatsuka, T. Katada, Y. Hirabayashi, K. Shimizu, J. Fujita-Yoshigaki, H. Sugiya, and S. Furuyama. 2001. Association of FcgammaRII with low-density detergent-resistant membranes is important for cross-linking-dependent initiation of the tyrosine phosphorylation pathway and superoxide generation. J. Immunol. 167:5814-5823.[Abstract/Free Full Text]
17
17. Kim, M. K., Z. Y. Huang, P. H. Hwang, B. A. Jones, N. Sato, S. Hunter, T. H. Kim-Han, R. G. Worth, Z. K. Indik, and A. D. Schreiber. 2003. Fcgamma receptor transmembrane domains: role in cell surface expression, gamma chain interaction, and phagocytosis. Blood 101:4479-4484.[Abstract/Free Full Text]
18
18. Kim, M. K., X. Q. Pan, Z. Y. Huang, S. Hunter, P. H. Hwang, Z. K. Indik, and A. D. Schreiber. 2001. Fc gamma receptors differ in their structural requirements for interaction with the tyrosine kinase Syk in the initial steps of signaling for phagocytosis. Clin. Immunol. 98:125-132.[CrossRef][Medline]
19
19. Kliks, S. C., A. Nisalak, W. E. Brandt, L. Wahl, and D. S. Burke. 1989. Antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever. Am. J. Trop. Med. Hyg. 40:444-451.[Abstract/Free Full Text]
20
20. Kontny, U., I. Kurane, and F. A. Ennis. 1988. Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells. J. Virol. 62:3928-3933.[Abstract/Free Full Text]
21
21. Kwiatkowska, K., and A. Sobota. 2001. The clustered Fcgamma receptor II is recruited to Lyn-containing membrane domains and undergoes phosphorylation in a cholesterol-dependent manner. Eur. J. Immunol. 31:989-998.[CrossRef][Medline]
22
22. Letourneur, O., I. C. Kennedy, A. T. Brini, J. R. Ortaldo, J. J. O'Shea, and J. P. Kinet. 1991. Characterization of the family of dimers associated with Fc receptors (Fc epsilon RI and Fc gamma RIII). J. Immunol. 147:2652-2656.[Abstract/Free Full Text]
23
23. Libraty, D. H., S. Pichyangkul, C. Ajariyakhajorn, T. P. Endy, and F. A. Ennis. 2001. Human dendritic cells are activated by dengue virus infection: enhancement by gamma interferon and implications for disease pathogenesis. J. Virol. 75:3501-3508.[Abstract/Free Full Text]
24
24. Littaua, R., I. Kurane, and F. A. Ennis. 1990. Human IgG Fc receptor II mediates antibody-dependent enhancement of dengue virus infection. J. Immunol. 144:3183-3186.[Abstract]
25
25. Lowry, M. B., A. M. Duchemin, J. M. Robinson, and C. L. Anderson. 1998. Functional separation of pseudopod extension and particle internalization during Fc gamma receptor-mediated phagocytosis. J. Exp. Med. 187:161-176.[Abstract/Free Full Text]
26
26. Mady, B. J., D. V. Erbe, I. Kurane, M. W. Fanger, and F. A. Ennis. 1991. Antibody-dependent enhancement of dengue virus infection mediated by bispecific antibodies against cell surface molecules other than Fc gamma receptors. J. Immunol. 147:3139-3144.[Abstract]
27
27. Maenaka, K., P. A. van der Merwe, D. I. Stuart, E. Y. Jones, and P. Sondermann. 2001. The human low affinity Fcgamma receptors IIa, IIb, and III bind IgG with fast kinetics and distinct thermodynamic properties. J. Biol. Chem. 276:44898-44904.[Abstract/Free Full Text]
28
28. Manes, S., G. del Real, and A. C. Martinez. 2003. Pathogens: raft hijackers. Nat. Rev. Immunol. 3:557-568.[CrossRef][Medline]
29
29. Martinez-Salas, E. 1999. Internal ribosome entry site biology and its use in expression vectors. Curr. Opin. Biotechnol. 10:458-464.[CrossRef][Medline]
30
30. Miller, K. L., A. M. Duchemin, and C. L. Anderson. 1996. A novel role for the Fc receptor gamma subunit: enhancement of Fc gamma R ligand affinity. J. Exp. Med. 183:2227-2233.[Abstract/Free Full Text]
31
31. Mitchell, M. A., M. M. Huang, P. Chien, Z. K. Indik, X. Q. Pan, and A. D. Schreiber. 1994. Substitutions and deletions in the cytoplasmic domain of the phagocytic receptor Fc gamma RIIA: effect on receptor tyrosine phosphorylation and phagocytosis. Blood 84:1753-1759.[Abstract/Free Full Text]
32
32. Morens, D. M., S. B. Halstead, P. M. Repik, R. Putvatana, and N. Raybourne. 1985. Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization. J. Clin. Microbiol. 22:250-254.[Abstract/Free Full Text]
33
33. Mukhopadhyay, S., J. Herre, G. D. Brown, and S. Gordon. 2004. The potential for Toll-like receptors to collaborate with other innate immune receptors. Immunology 112:521-530.[CrossRef][Medline]
34
34. Nimmerjahn, F., and J. V. Ravetch. 2006. Fcgamma receptors: old friends and new family members. Immunity 24:19-28.[CrossRef][Medline]
35
35. Odin, J. A., J. C. Edberg, C. J. Painter, R. P. Kimberly, and J. C. Unkeless. 1991. Regulation of phagocytosis and [Ca2+]i flux by distinct regions of an Fc receptor. Science 254:1785-1788.[Abstract/Free Full Text]
36
36. Ortiz-Stern, A., and C. Rosales. 2003. Cross-talk between Fc receptors and integrins. Immunol. Lett. 90:137-143.[CrossRef][Medline]
37
37. Parren, P. W., and D. R. Burton. 2001. The antiviral activity of antibodies in vitro and in vivo. Adv. Immunol. 77:195-262.[Medline]
38
38. Peiris, J. S., and J. S. Porterfield. 1981. Antibody-dependent enhancement of plaque formation on cell lines of macrophage origin—a sensitive assay for antiviral antibody. J. Gen. Virol. 57:119-125.[Abstract/Free Full Text]
39
39. Rabinovitch, M. 1995. Professional and non-professional phagocytes: an introduction. Trends Cell Biol. 5:85-87.[CrossRef][Medline]
40
40. Ravetch, J. V., and S. Bolland. 2001. IgG Fc receptors. Annu. Rev. Immunol. 19:275-290.[CrossRef][Medline]
41
41. Sabin, A. 1952. Research on dengue during World War II. Am. Trop. Med. Hyg. 1:30-50.[Abstract/Free Full Text]
42
42. Schlesinger, J. J., and S. E. Chapman. 1999. Influence of the human high-affinity IgG receptor FcgammaRI (CD64) on residual infectivity of neutralized dengue virus. Virology 260:84-88.[CrossRef][Medline]
43
43. Simons, K., and D. Toomre. 2000. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1:31-39.[CrossRef][Medline]
44
44. Smith, A. E., and A. Helenius. 2004. How viruses enter animal cells. Science 304:237-242.[Abstract/Free Full Text]
45
45. Takai, T. 2002. Roles of Fc receptors in autoimmunity. Nat. Rev. Immunol. 2:580-592.[Medline]
46
46. Takai, T., M. Li, D. Sylvestre, R. Clynes, and J. V. Ravetch. 1994. FcR gamma chain deletion results in pleiotrophic effector cell defects. Cell 76:519-529.[CrossRef][Medline]
47
47. Thein, S., J. Aaskov, T. T. Myint, T. N. Shwe, T. T. Saw, and A. Zaw. 1993. Changes in levels of anti-dengue virus IgG subclasses in patients with disease of varying severity. J. Med. Virol. 40:102-106.[Medline]
48
48. Van den Herik-Oudijk, I. E., P. J. Capel, T. van der Bruggen, and J. G. Van de Winkel. 1995. Identification of signaling motifs within human Fc gamma RIIa and Fc gamma RIIb isoforms. Blood 85:2202-2211.[Abstract/Free Full Text]
49
49. Van den Herik-Oudijk, I. E., M. W. Ter Bekke, M. J. Tempelman, P. J. Capel, and J. G. Van de Winkel. 1995. Functional differences between two Fc receptor ITAM signaling motifs. Blood 86:3302-3307.[Abstract/Free Full Text]
50
50. Van den Herik-Oudijk, I. E., N. A. Westerdaal, N. V. Henriquez, P. J. Capel, and J. G. Van De Winkel. 1994. Functional analysis of human Fc gamma RII (CD32) isoforms expressed in B lymphocytes. J. Immunol. 152:574-585.[Abstract]
51
51. van de Winkel, J. G., and C. L. Anderson. 1991. Biology of human immunoglobulin G Fc receptors. J. Leukoc. Biol. 49:511-524.[Medline]
52
52. van Vugt, M. J., A. F. Heijnen, P. J. Capel, S. Y. Park, C. Ra, T. Saito, J. S. Verbeek, and J. G. van de Winkel. 1996. FcR gamma-chain is essential for both surface expression and function of human Fc gamma RI (CD64) in vivo. Blood 87:3593-3599.[Abstract/Free Full Text]
53
53. Vaughn, D. W., S. Green, S. Kalayanarooj, B. L. Innis, S. Nimmannitya, S. Suntayakorn, T. P. Endy, B. Raengsakulrach, A. L. Rothman, F. A. Ennis, and A. Nisalak. 2000. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J. Infect. Dis. 181:2-9.[CrossRef][Medline]
54
54. Warmerdam, P. A., P. W. Parren, A. Vlug, L. A. Aarden, J. G. van de Winkel, and P. J. Capel. 1992. Polymorphism of the human Fc gamma receptor II (CD32): molecular basis and functional aspects. Immunobiology 185:175-182.[Medline]
55
55. Wellington, M., J. M. Bliss, and C. G. Haidaris. 2003. Enhanced phagocytosis of Candida species mediated by opsonization with a recombinant human antibody single-chain variable fragment. Infect. Immun. 71:7228-7231.[Abstract/Free Full Text]
56
56. Woof, J. M., and D. R. Burton. 2004. Human antibody-Fc receptor interactions illuminated by crystal structures. Nat. Rev. Immunol. 4:89-99.[CrossRef][Medline]
57
57. Wu, S. J., G. Grouard-Vogel, W. Sun, J. R. Mascola, E. Brachtel, R. Putvatana, M. K. Louder, L. Filgueira, M. A. Marovich, H. K. Wong, A. Blauvelt, G. S. Murphy, M. L. Robb, B. L. Innes, D. L. Birx, C. G. Hayes, and S. S. Frankel. 2000. Human skin Langerhans cells are targets of dengue virus infection. Nat. Med. 6:816-820.[CrossRef][Medline]

---

Journal of Virology, October 2006, p. 10128-10138, Vol. 80, No. 20
0022-538X/06/$08.00+0     doi:10.1128/JVI.00792-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Perez, L. G., Costa, M. R., Todd, C. A., Haynes, B. F., Montefiori, D. C. (2009). Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41. J. Virol. 83: 7397-7410 [Abstract] [Full Text]  
• Rodrigo, W. W. I. S., Alcena, D. C., Kou, Z., Kochel, T. J., Porter, K. R., Comach, G., Rose, R. C., Jin, X., Schlesinger, J. J. (2009). Difference between the Abilities of Human Fc{gamma} Receptor-Expressing CV-1 Cells To Neutralize American and Asian Genotypes of Dengue Virus 2. CVI 16: 285-287 [Abstract] [Full Text]  
• Shanaka, W. W., Rodrigo, I., Alcena, D. C., Rose, R. C., Jin, X., Schlesinger, J. J. (2009). An Automated Dengue Virus Microneutralization Plaque Assay Performed in Human Fc{gamma} Receptor-expressing CV-1 Cells. Am J Trop Med Hyg 80: 61-65 [Abstract] [Full Text]  
• Saavedra, M. T., Hughes, G. J., Sanders, L. A., Carr, M., Rodman, D. M., Coldren, C. D., Geraci, M. W., Sagel, S. D., Accurso, F. J., West, J., Nick, J. A. (2008). Circulating RNA Transcripts Identify Therapeutic Response in Cystic Fibrosis Lung Disease. Am. J. Respir. Crit. Care Med. 178: 929-938 [Abstract] [Full Text]  
• Boonnak, K., Slike, B. M., Burgess, T. H., Mason, R. M., Wu, S.-J., Sun, P., Porter, K., Rudiman, I. F., Yuwono, D., Puthavathana, P., Marovich, M. A. (2008). Role of Dendritic Cells in Antibody-Dependent Enhancement of Dengue Virus Infection. J. Virol. 82: 3939-3951 [Abstract] [Full Text]  
• Blackley, S., Kou, Z., Chen, H., Quinn, M., Rose, R. C., Schlesinger, J. J., Coppage, M., Jin, X. (2007). Primary Human Splenic Macrophages, but Not T or B Cells, Are the Principal Target Cells for Dengue Virus Infection In Vitro. J. Virol. 81: 13325-13334 [Abstract] [Full Text]  
• Goncalvez, A. P., Engle, R. E., St. Claire, M., Purcell, R. H., Lai, C.-J. (2007). Monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention. Proc. Natl. Acad. Sci. USA 104: 9422-9427 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rodrigo, W. W. S. I. Articles by Schlesinger, J. J. Search for Related Content PubMed PubMed Citation Articles by Rodrigo, W. W. S. I. Articles by Schlesinger, J. J.