Fc␥ receptor (Fc␥R)-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. Fc␥RIA (CD64) and Fc␥RIIA (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 Fc␥RIA and constitutively in Fc␥RIIA is required for phagocytosis mediated by these receptors. To determine whether Fc␥RIA and Fc␥RIIA 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 Fc␥RIIA appeared to do so far more effectively. Abrogation of Fc␥RIA 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 Fc␥RIIA 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 Fc␥RIA and Fc␥RIIA with respect to their immune-enhancing capabilities and suggest that different mechanisms of dengue virus immune complex internalization may operate between these Fc␥Rs.

Understanding the pathogenesis of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) requires the precise identification of dengue virus (DV)-permissive target cells. In a previous study using unfractionated human peripheral blood mononuclear cells, we found that monocytes, but not B or T cells, were the principal DV-permissive cells in the absence of DV-immune pooled human sera (PHS) and the major mediators of antibody-dependent enhancement in the presence of PHS. To further identify DV-permissive target cells in other tissues and organs, we isolated human splenic mononuclear cells (MNCs), inoculated them with DV type 2 (strain 16681) in the presence or absence of PHS, and assessed their infection either directly using flow cytometry and reverse transcription-PCR (RT-PCR) assays or indirectly by plaque assay. We found that in the absence of PHS, a small proportion of splenic macrophages appeared to be positive for DV antigens in comparison to staining controls by the flow cytometric assay (0.77% ؎ 1.00% versus 0.18% ؎ 0.12%; P ‫؍‬ 0.07) and that viral RNA was detectable by the RT-PCR assay in MNCs exposed to DV. Additionally, supernatants from cultures of DV-exposed MNCs contained infectious virions that were readily detectable by plaque assay. The magnitude of infection was significantly enhanced in splenic macrophages in the presence of highly diluted PHS (5.41% ؎ 3.53% versus 0.77% ؎ 1.00%; P ‫؍‬ 0.001). In contrast, primary T and B cells were not infected in either the presence or absence of PHS. These results provide evidence, for the first time, that human primary splenic macrophages, rather than B or T cells, are the principal DV-permissive cells in the spleen and that they may be uniquely important in the initial steps of immune enhancement that leads to DHF/DSS in some DV-infected individuals.