Interleukin-1 beta-induced up-regulation of opioid receptors in the untreated and morphine-desensitized U87 MG human astrocytoma cells

Abstract

Background: Interleukin-1beta (IL-1β) is a pro-inflammatory cytokine that can be produced in the central nervous system during inflammatory conditions. We have previously shown that IL-1β expression is altered in the rat brain during a morphine tolerant state, indicating that this cytokine may serve as a convergent point between the immune challenge and opiate mediated biological pathways. We hypothesized that IL-1β up-regulates opioid receptors in human astrocytes in both untreated and morphine-desensitized states.

Methods: To test this hypothesis, we compared the basal expression of the mu (MOR), delta (DOR), and kappa (KOR) opioid receptors in the human U87 MG astrocytic cell line to SH-SY5Y neuronal and HL-60 immune cells using absolute quantitative real time RT-PCR (AQ-rt-RT-PCR). To demonstrate that IL-1β induced up-regulation of the MOR, DOR and KOR, U87 MG cells (2 x 105 cells/well) were treated with IL-1β (20 ng/mL or 40 ng/mL), followed by co-treatment with interleukin-1 receptor antagonist protein (IL-1RAP) (400 ng/mL or 400 ng/mL). The above experiment was repeated in the cells desensitized with morphine, where U87 MG cells were pre-treated with 100 nM morphine. The functionality of the MOR in U87 MG cells was then demonstrated using morphine inhibition of forksolin-induced intracellular cAMP, as determined by radioimmunoassay.

Results: U87 MG cells treated with IL-1β for 12 h showed a significant up-regulation of MOR and KOR. DOR expression was also elevated, although not significantly. Treatment with IL-1β also showed a significant up-regulation of the MOR in U87 MG cells desensitized with morphine. Co-treatment with IL-1β and interleukin-1 receptor antagonist protein (IL-1RAP) resulted in a significant decrease in IL-1β-mediated MOR up-regulation.

Conclusion: Our results indicate that the pro-inflammatory cytokine, IL-1β, affects opiate-dependent pathways by up-regulating the expression of the MOR in both untreated and morphine-desensitized U87 MG.

Figure 1

Basal levels of MOR, DOR and KOR mRNA in neuronal and immune cell lines. Basal levels (copy number) of the human MOR, DOR and KOR in the U87 MG astrocytic, HL-60 (TPA differentiated and undifferentiated), NMB, and SH-SY5Y cell lines were determined using absolute quantitative real time RT-PCR (AQ-rt-RT-PCR). GAPDH was used to normalize the levels in each cell line. Data are indicated as the mean ± SE.

Figure 2

Immunofluorescence staining of the human MOR. Immunofluorescence staining was used to visualize the basal expression level of the human MOR. (A) U87 MG cells stained with goat anti-rabbit IgG (1:1,000) alone (negative control); (B) U87 MG cells stained with 4'-6-diamidino-2-phenylindole (DAPI) (1:1,000); (C and E) U87 MG cells stained with rabbit anti-MOR (1:1,000); (D) A superimposed image of B and C to show location of the MOR.

Figure 3

The effects of morphine,naloxone,and endomorphin-1 and endomorphin-2,on forskolin-induced cAMP levels. Functionality of the MOR expressed in U87 MG cells was determined using a forskolin-induced cAMP accumulation assay. (A) cAMP accumulation levels were determined in basal (untreated) U87 MG cells and in U87 MG cells treated with forskolin alone (75 μM), morphine alone (10 μM), forskolin (75 μM) + morphine (10 μM), or forskolin (75 μM) + morphine (10 μM) + naloxone (10 μM); (B) cAMP accumulation levels were determined in basal (untreated) U87 MG cells and in U87 MG cells treated with forskolin alone (75 μM), endomorphin-1 alone (10 μM), endomorphin-2 alone (10 μM), fosrkolin (75 μM) + endomorphin-1 (10 μM), forskolin (75 μM) + endomorphin-2 (10 μM), forskolin (75 μM) + endomorphin-1 (10 μM) + naloxone (10 μM), or forskolin (75 μM ) + endomorphin-2 (10 μM) + naloxone (10 μM). Data are the mean ± SE. A one-way ANOVA was used to determine significance. *P <0.05 compared to basal treatment; ^P <0.05 compared to forskolin treatment alone.

Figure 4

The effects of IL-1βon opioid expression in U87 MG cells. U87 MG cells were treated with either cell culture medium (control) or IL-1β (20 ng/mL or 40 ng/mL) for 12 h. Real time RT-PCR was used to determine the levels of MOR (A), DOR (B), and KOR (C); GAPDH was used to normalize the receptor levels. Data are the mean ± SE. A Student’s t-test was used to determine significance. *P <0.05 compared to control cells.

Figure 5

The effects of IL-1RAP on IL-1β-induced up-regulation of the MOR in U87 MG cells. U87 MG cells were treated with medium (control), IL-1β (20 ng/mL), IL-1RAP (400 ng/mL) + vehicle, IL-1RAP (400 ng/mL) + IL-1β (20 ng/mL), IL-1RAP (4,000 ng/mL) + vehicle, or IL-1RAP (4,000 ng/mL) + IL-1β (20 ng/mL) for 12 h. Real time RT-PCR was used to determine the levels of the MOR; GAPDH was used to normalize the MOR levels. Data are the mean ± SE. A Student t-test was used to determine significance. *P <0.05 compared to control; ^P <0.001 compared to IL-1β (alone); ^#P <0.01 compared to IL-1β (alone).

Figure 6

The effects of morphine on MOR expression in U87 MG cells. U87 MG cells were treated with either vehicle (cell culture medium) or morphine (100 nM) for 0 (control), 45 minutes, 3, 6, 12, 24, or 48 hours. Real time RT-PCR was used to determine the copy number of the MOR and GAPDH. GAPDH levels were used to normalize the MOR levels. Each time-point was adjusted by the appropriate time-point control. Data are the mean ± SE. A Student’s t-test was used to determine significance. *P <0.05 compared to control.

Figure 7

The effects of morphine pre-treatment on IL-1β-induced up-regulation of the MOR in U87 MG cells. U87 MG cells were pre-treated with either vehicle (control) or morphine (100 nM) for 24 h. Following pre-treatment, the medium was aspirated and fresh cell culture medium containing either vehicle or IL-1β (20 ng/mL) was added and the cells were incubated for 12 h. Absolute quantitative real time RT-PCR (AQ-rt-RT-PCR) was used to determine expression of the MOR and GAPDH. Levels of MOR were normalized to GAPDH. Data are the mean ± SE. A Student’s t-test was used to determine significance. *P <0.05 compared to the control; ^#P <0.05 compared to IL-1β (alone); ^P <0.001 compared to the control.