Expression of the mu opioid receptor in the human immunodeficiency virus type 1 transgenic rat model

Abstract

Opioids, via the mu opioid receptor (MOR), can exacerbate bacterial infections and the immunopathogenesis of human immunodeficiency virus type 1 (HIV-1) infection. Recently, an HIV-1 transgenic (HIV-1Tg) rat model containing circulating HIV-1 gp120 was created. Using real-time reverse transcription-PCR, we found that MOR mRNA levels were significantly higher in the peritoneal macrophages of the HIV-1Tg rat than those in control animals. Lipopolysaccharide, a bacterial endotoxin, induced secretion of the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-beta (IL-beta), and IL-10 in the HIV-1Tg rat and further increased MOR expression. Ex vivo studies showed that MOR expression was up-regulated in the peritoneal macrophages of F344 control rats by exposure to serum from HIV-1Tg rats and that MOR up-regulation was abolished by addition of gp120 antibody to the serum. In human TPA-differentiated HL-60 cells, which are macrophage-like cells, LPS-induced MOR mRNA up-regulation was greater in gp120-pretreated cells than in vehicle-pretreated cells. Our data suggest that in individuals infected with HIV-1, the MOR is up-regulated, possibly by circulating HIV-1 proteins such as gp120, and HIV-1 proteins may play a significant role in modulating the response to bacterial infection in opioid-using HIV-infected individuals. Furthermore, our results demonstrate that the new HIV-1Tg rat model can be a valuable tool with which to study MOR gene expression and its effects in the continuous presence of HIV viral proteins.

FIG. 1.

MOR mRNA levels in peritoneal macrophages of HIV-1Tg rats. MOR mRNA levels in the peritoneal macrophages from HIV-1Tg and F344 rats were compared using real-time RT-PCR (A and B). Delta Rn refers to the delta reaction that is the normalized intensity of fluorescence emission from the reporter dye for MOR while MOR is amplified as a function of the reaction cycle (Ct). Real-time RT-PCR of the cDNA from the peritoneal macrophages was run an additional 7 cycles after crossing the amplification threshold, and the PCR products were electrophoresed on a 3% agarose gel (C). n = 3; *, P < 0.05 compared to F344 control rats.

FIG. 2.

LPS-induced serum levels of TNF-α, IL-1β, and IL-10 in HIV-1Tg rats. Serum levels of TNF-α, IL-1β, and IL-10 from HIV-1Tg and F344 rats were measured by ELISA 2 h following an i.p. injection with either saline or LPS (250 μg/kg). n = 3; *, P < 0.05 compared to saline treatment; ^, P < 0.05 compared to saline-treated F344 rats.

FIG. 3.

LPS-induced MOR expression in peritoneal macrophages of HIV-1Tg rats. HIV-1Tg and F344 control rats were injected (i.p.) with either saline or LPS (1.25 mg/kg). Twenty-four hours after treatment, the animals were sacrificed and the peritoneal macrophages were isolated for analysis of MOR mRNA by real-time RT-PCR. n = 3; *, P < 0.05 compared to F344 rats given saline; ^, P < 0.05 compared to F344 rats given LPS.

FIG. 4.

Effects of HIV-1Tg serum on MOR expression in F344 rats. Peritoneal macrophages were isolated from six F344 control rats, and treated with either heated HIV-1Tg rat serum (20%), recombinant gp120 protein (1 nM), LPS (10 μg/ml), gp120 monoclonal antibody alone, heated HIV-1Tg rat serum (20%) plus gp120 monoclonal antibody, or control vehicle (culture medium) for 18 h at 37°C. The cells were harvested for total RNA isolation, and MOR mRNA levels were determined by real-time RT-PCR analysis. n = 3; *, P < 0.05 compared to control vehicle.

FIG. 5.

Effects of pretreatment with gp120 protein on LPS-induced up-regulation of MOR mRNA expression in TPA-HL-60 cells. TPA-HL-60 cells were pretreated with either vehicle medium or 1 nM recombinant gp120 protein for 24 h and then treated for an additional 12 h with control vehicle (culture medium) or different concentrations of LPS (10 ng/ml or 100 ng/ml). The effects of gp120 pretreatment on LPS-induced up-regulation of MOR expression were then examined by real-time RT-PCR. n = 3; *, P < 0.05 compared to control vehicle; ^, P < 0.05 compared to treatment with vehicle followed by LPS at 10 ng/ml and 100 ng/ml, respectively.