Use of Colloidal Silica (Sepracell-MN) for Enrichment of Dendritic Cells From Human Peripheral Blood: Comparison With Other Methods

Three methods are described for enrichment of dendritic cells from human peripheral blood. In method 1 , mononuclear cells were incubated in plastic tissue culture flasks for two h. Nonadherent cells were removed. Adherent cells were washed to remove floating cells and incubated for 14 h at 37#{176in}C 5% CO2. Carbonyb iron was added, and the flasks were incubated for another 2 h. Nonadherent cells were subjected to centrifugation over metrizamide gradient. Phagocytic cells containing ingested carbonyl iron, small lymphocytes, and free carbonyl iron particles passed through the metrizamide, while the interface cell population was enriched for dendritic cells. The purity and yield of enriched dendritic cells were 52.8% and 0.05%, respectively. In method 2, adherent mononuclear cells were cultured overnight, and the released cells (released adherent cells) were centrifuged over metrizamide to separate low-density cells. Monocytes from this low-density cell population were removed by panning over human gamma globulin-coated plastic Petri dishes. In this method the average purity and yield of DC were 63.8% and 0.1%, respectively. In method 3, released adherent cells were treated with anti-CD5 and anti- CDI4 monoclonal antibodies plus baby rabbit complement for 15 mm, washed, and centrifuged with colloidal silica (Sepracell-MN). Centrifugation with Sepracell-MN was repeated three times. Low-density cells were panned twice over human gamma globulincoated plastic Petri dishes. Nonadherent cells were highly enriched for DC. Contamination of T cells, B cells, and NK cells was undetectable by flow cytofluorometry. Contamination of monocytes was 85.0% purity and 0.4% yield. This method (method 3) combines adherence, complement-dependent lysis, centrifugation with colloidal silica, and panning and provides the best yield and purity; it is therefore recommended for optimal purification of DC

CD4-independent infection of human peripheral blood dendritic cells with isolates of human immunodeficiency virus type 1

Dendritic cells (DC) are members of a distinct family of bone marrow-derived leukocytes. DC are potent accessory cells for a number of T cell-mediated immune responses, including autologous and allogeneic mixed leukocyte reactions, and mitogen- and antigenstimulated lymphocyte proliferation. In the present study, DC purified from human peripheral blood were inoculated with various strains (IIIB, SF2, WMJ1, SF162, 89.6 and clone HXB2) of human immunodeficiency virus type 1 (HIV-1) displaying different patterns of cellular tropism. Viral replication was demonstrated by detection of p24 antigen (Ag) intracellularly and in culture supernatants, and by Southern and Northern blot analyses for the presence of HIV DNA and RNA, respectively, within infected cells. Cellfree and cell-associated p24 Ag levels rose substantially when DC were inoculated with strains SF162, 89.6 and clone HXB2. In contrast, p24 Ag levels rose only marginally after inoculation of DC with strains IIIB, SF2 and WMJ1. Purified DC did not express detectable membrane CD4, although CD4 mRNA was detected by reverse transcriptase PCR. The presence of anti-CD4 monoclonal antibodies failed to block infection of DC by any of the HIV strains tested, suggesting the existence of a CD4-independent alternative pathway of viral entry. The possibility that DC serve as a reservoir for HIV-1 must be considered

In Vitro Infection of Natural Killer Cells with Different Human Immunodeficiency Virus Type 1 Isolates

Natural killer (NK) cells are a discrete subset of leukocytes, distinct from T and B lymphocytes. NK cells mediate spontaneous non-MHC-restricted killing of a wide variety of target cells without prior sensitization and appear to be involved in initial protection against certain viral infections. Depressed NK cell-mediated cytotoxicity, one of the many immunological defects observed in AIDS patients, may contribute to secondary virus infections. Here we report that clonal and purified polyclonal populations of NK cells, which expressed neither surface CD4 nor CD4 mRNA, were susceptible to infection with various isolates of human immunodeficiency virus type 1 (HIV-1). Viral replication was demonstrated by detection of p24 antigen intracellularly and in culture supernatants, by the presence of HIV DNA within infected cells, and by the ability of supernatants derived from HIV-infected NK cells to infect peripheral blood mononuclear cells or CD4+ cell lines. Infection of NK cells was not blocked by anti-CD4 or anti-FcyRIII monoclonal antibodies. NK cells from HIV-infected and uninfected cultures were similar in their ability to lyse three different target cells. Considerable numbers of cells died in HIV-infected NK cell cultures. These results suggest that loss of NK cells in AIDS patients is a direct effect of HIV infection but that reduced NK cell function involves another mechanism. The possibility that NK cells serve as a potential reservoir for HIV-1 must be considere