HIV-1 gp120 primes lymphocytes for opioid-induced, beta-arrestin 2-dependent apoptosis

Abstract

The mechanisms by which opioids affect progression of human immunodeficiency virus type 1 (HIV-1) infection are not well-defined. HIV-1 gp120 is important in the apoptotic death of uninfected, bystander T cells. In this study, we show that co-treatment of human peripheral blood mononuclear cells (PBMC) with HIV-1 gp120/morphine synergistically induces apoptosis in PBMC. Co-treatment of murine splenocytes from mu opiate receptor knockout mice with gp120/morphine resulted in decreased apoptosis when compared to splenocytes from wild type mice. Co-treatment of human PBMC or murine splenocytes with gp120/morphine led to decreased expression of beta-arrestin 2, a protein required for opioid-mediated signaling. The role of beta-arrestin 2 was confirmed in Jurkat lymphocytes, in which 1) over-expression of beta-arrestin 2 inhibited gp120/morphine-induced apoptosis and 2) RNA interference of beta-arrestin 2 expression enhanced gp120/morphine-induced apoptosis. These data suggest a novel mechanism by which HIV-1 gp120 and opioids induce lymphocyte cell death.

Fig. 1

Effect of gp120/anti-gp120 and morphine on lymphocyte apoptosis. gp120/anti-gp120 and morphine induces apoptosis in human PBMC. PBMC were incubated with gp120 (150 ng/ml) for 1 h at 4 °C, followed by addition of polyclonal anti-gp120 (150 ng/ml) as described. After 1 h incubation at 37 °C, morphine sulfate (3 μM) was added. After 24 h, cell apoptosis was assayed by flow cytometry after staining with propidium iodide. *p <0.01 vs control, morphine, and gp120/anti-gp120. **p <0.01 vs morphine and gp120/anti-gp120.

Fig. 2

gp120/anti-gp120 and morphine-induced apoptosis in splenocytes occurs through the μ opioid receptor. Splenocytes from wild type and μOR knockout mice were treated with gp120/anti-gp120 and morphine as above. Splenocytes were collected and cell apoptosis determined as above. *p <0.01 vs a combination of gp120/anti-gp120 and morphine in splenocytes from wild type mice.

Fig. 3

gp120/morphine-mediated apoptosis is mediated by β-arrestin 2 expression (a). gp120/morphine reduces β-arrestin 2 expression. Freshly isolated human PBMC were treated with gp120 (150 ng/ml) for 1 h at 4 °C, followed by addition of polyclonal anti-gp120 (150 ng/ml). After 1 h at 37 °C, PBMC were treated with 3 μM morphine sulfate for 2 h and the expression of β-arrestin 2 was determined by real time quantitative RT PCR. *p<0.01 vs control, morphine, and gp120/anti-gp120. (b). gp120/morphine-induced apoptosis is inhibited by over-expression of β-arrestin 2. Jurkat T cells transfected with pcDNA3-GFP or pcDNA3/β-arrestin 2 were incubated for 16 h and then treated with gp120 (150 ng/ml) for 1 h at 4 °C, followed by the addition of polyclonal anti-gp120 (150 ng/ml). After 1 h at 37 °C, cells were treated with 10 μM morphine sulfate for an additional 36 h and the percent apoptosis was determined by flow cytometric analysis. Mean values were derived from three independent experiments. *p <0.01 compared to transfected pcDNA3-GFP.

Fig. 4

Inhibition of β-arrestin 2 expression by RNAi enhances gp120/morphine-induced apoptosis.(a). Suppression of endogenously-expressed β-arrestin 2 using RNA interference. Jurkat T cells were transfected with 50 nM control RNAi or 50 nM β-arrestin 2 RNAi for 24 h. The expression of β-arrestin 2 was determined by real time quantitative PCR. (b). Apoptotic response of lymphocytes to gp120/anti-gp120 and morphine following β-arrestin 2 RNA interference. Jurkat T cells were transfected with GFP or β-arrestin 2 RNAi plasmids or control vector, cells treated with gp120/anti-gp120 and morphine, and analysis of apoptosis performed as described above. *p <0.01 compared with transfected control RNAi.

Fig. 5

gp120/morphine-induced apoptosis in splenocytes requires β-arrestin 2. Splenocytes from wild type mice and β-arrestin 2 knockout mice were treated with gp120/anti-gp120 and morphine as in a. Cell apoptosis was determined as in a. *p <0.01 vs. a combination of gp120/anti-gp120 and morphine in splenocytes from wild type mice.