Gamma interferon primes productive human immunodeficiency virus infection in astrocytes

Abstract

Considerable controversy exists over whether astrocytes can support human immunodeficiency virus (HIV) infection. We evaluated the impact of three cytokines critical to the development of HIV neuropathogenesis, gamma interferon (IFN-gamma), granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha, on priming astrocytes for HIV infection. We demonstrate that IFN-gamma was the most potent in its ability to facilitate substantial productive HIV infection of an astroglioma cell line (U87MG) and human fetal astrocytes (HFA). The mechanism of IFN-gamma-mediated priming of HIV in HFA is unlikely to be at the level of up-regulation of receptors and coreceptors relevant to HIV entry. These data demonstrate that cytokine priming can alter HIV replication in astrocytes.

FIG. 1.

Purity of HFA cultures. Flow cytometry was performed to evaluate the purity of the HFA cultures. Cells were stained for an astrocyte marker (glial fibrillary acidic protein [GFAP]) (A, B), a microglia marker (CD68) (A, C), an astrocyte precursor marker (nestin) (B), or a neuron marker (MAP2) (C). Dot plots shown are representative of the typical purity of HFA cultures used throughout this study. APC, allophycocyanin; PE, phycoerythrin; FITC, fluorescein isothiocyanate.

FIG. 2.

Cytokine receptor expression on astrocytes. U87MG cells (A) or HFA (B) were left untreated or treated with GM-CSF (50 ng/ml), IFN-γ (100 ng/ml), or TNF-α (100 ng/ml). Cytokine concentrations were based on the concentrations determined to induce maximal HIV expression, except for TNF-α, which did not induce HIV expression (Fig. 3). Percentages of the expression of the GM-CSF receptor (R), IFN-γ receptor, and TNF-α receptor was measured 24 h poststimulation by flow cytometry. Data are representative of three experiments. Error bars represent standard deviations of the means.

FIG. 3.

Effects of GM-CSF and IFN-γ prestimulation on HIV replication in astrocytes. U87MG cells (A) and HFA (B) were prestimulated with increasing doses of either GM-CSF or IFN-γ (IFN-g) for 24 h followed by HIV BAL infection for 24 h. HIV replication was determined by measuring HIV p24 levels by enzyme-linked immunosorbent assay 7 days postinfection. The dotted line represents the basal level of HIV infection of astrocytes without cytokine pretreatment. Data are representative of three experiments performed in quadruplicate. Error bar represent standard deviations of the means. Asterisks denote significant values (P < 0.05) in comparison to values for untreated cultures as evaluated by analysis of variance.

FIG. 4.

Effect of GM-CSF and IFN-γ on the expression of receptors and chemokine coreceptors relevant to HIV replication. U87MG cells (A) and HFA (B) were left untreated or treated with GM-CSF (50 ng/ml) or IFN-γ (100 ng/ml) for 24 h. Levels of expression of receptors on the x axis were measured by flow cytometry. Isotype refers to mouse immunoglobulin G. Data are representative of three experiments. Error bars represent standard deviations of the means. hMR, human mannose receptor; IFN-g, gamma IFN.