HIV-1 viral envelope glycoprotein gp120 triggers an inflammatory response in cultured rat astrocytes and regulates the functional expression of P-glycoprotein

In this work, we examined the ability of gp120, a human immunodeficiency virus-1 (HIV-1) viral envelope glycoprotein, to trigger the innate immune response in astrocytes, an HIV-1 brain cellular target, and we investigated the functional expression of the ATP-binding cassette membrane transporter P-glycoprotein (P-gp) in primary cultures of rat astrocytes treated with gp120 or cytokines [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6]. Standard 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium and D-mannitol uptake assays confirmed that HIV-1 _96ZM651 gp120 treatment did not alter cell viability or membrane permeability. Semiquantitative reverse-transcriptase polymerase chain reaction analysis and enzyme-linked immunosorbent assay demonstrated increased TNF-α, IL-1β, and IL-6 mRNA and protein expression in cultures treated with HIV-1_96ZM651 gp120, suggesting in vitro activation of immune responses. Cytokine secretion was detected when CXCR4 but not CCR5 was inhibited with a specific antibody, implying that cytokine secretion is primarily mediated via CCR5 in astrocytes triggered with HIV-1_96ZM651 gp120. P-gp protein expression was increased in astrocyte cultures exposed to TNF-α (2.9-fold) or IL-1β (1.6-fold) but was decreased profoundly in the presence of IL-6 (8.9-fold), suggesting that IL-6 is primarily involved in modulating P-gp expression. In parallel, after HIV-1_96ZM651 gp120 treatment, immunoblotting analysis showed a significant decrease in P-gp expression (4.7-fold). Furthermore, the accumulation of two P-gp substrates, digoxin and saquinavir (an HIV-1 protease inhibitor), was enhanced (1.5- to 1.8-fold) in HIV-1_96ZM651 gp120-treated astrocyte monolayers but was not altered by P-gp inhibitors [e.g., valspodar (PSC833) and elacridar (GF120918)], suggesting a loss of transport activity. Taken together, these data imply that HIV-1_96ZM651 gp120 or cytokine treatment modulate P-gp functional expression in astrocytes, which may lead to complex drug-transporter interactions during HIV-1 encephalitis-associated immune responses.