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

Byrne LS, Peng J, Sarkar S, Chang SL - J Neuroinflammation (2012)

Bottom Line: DOR expression was also elevated, although not significantly.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of NeuroImmune Pharmacology, Seton Hall University, 400 South Orange Ave, South Orange, NJ 07079, USA.

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.

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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.
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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.

Mentions: Opioid receptor expression in the U87 MG astrocytoma cell line was compared to TPA-differentiated and undifferentiated HL-60 immune cells and SH-SY5Y and NMB neuronal cells. All of the cell lines examined expressed all three opioid receptors - MOR, DOR and KOR - although the levels of expression of each receptor varied in different cell lines. SH-SY5Y cells had the highest MOR expression (1.63 x 107 ± 0.41 x 107 copies of MOR/μg of total RNA) compared to the TPA-differentiated HL-60 cells (6.06 x 105 ± 0.30 x 105 copies of MOR/μg of total RNA), undifferentiated HL-60 cells (1.43 x 105 ± 0.67 x 105 copies of MOR/μg of total RNA), NMB cells (9.5 x 104 ± 4.8 x 104 copies of MOR/μg of total RNA), and U87 MG cells (3.43 x 105 ± 0.67 x 105 copies of MOR/μg of total RNA). NMB cells expressed the highest levels of DOR (3.68 x 107 ± 0.48 x 107 copies of DOR/μg of total RNA) compared to the TPA-differentiated HL-60 cells (1.94 x 106 ± 0.3 x 106 copies of DOR/μg of total RNA), undifferentiated HL-60 cells (1.26 x 106 ± 0.06 x 106 copies of DOR/μg of total RNA), SH-SY5Y cells (1.67 x 107 ± 0.41 x 107 copies of DOR/μg of total RNA), and U87 MG cells (7.86 x 105 ± 0.06 x 105 copies of DOR/μg of total RNA). TPA-differentiated HL-60 cells had the highest KOR expression (2.18 x 107 ± 0.30 x 107 copies of KOR/μg of total RNA) compared to HL-60 cells (5.55 x 106 ± 0.14 x 106 copies of DOR/μg of total RNA), NMB cells (1.17 x 107 ± 0.48 x 107 copies of DOR/μg of total RNA), SH-SY5Y cells (2.25 x 104 ± 0.32 x 104 copies of DOR/μg of total RNA), and U87 MG cells (1.21 x 105 ± 0.13 x 105 copies of DOR/μg of total RNA) (Figure 1).


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

Byrne LS, Peng J, Sarkar S, Chang SL - J Neuroinflammation (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526549&req=5

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.
Mentions: Opioid receptor expression in the U87 MG astrocytoma cell line was compared to TPA-differentiated and undifferentiated HL-60 immune cells and SH-SY5Y and NMB neuronal cells. All of the cell lines examined expressed all three opioid receptors - MOR, DOR and KOR - although the levels of expression of each receptor varied in different cell lines. SH-SY5Y cells had the highest MOR expression (1.63 x 107 ± 0.41 x 107 copies of MOR/μg of total RNA) compared to the TPA-differentiated HL-60 cells (6.06 x 105 ± 0.30 x 105 copies of MOR/μg of total RNA), undifferentiated HL-60 cells (1.43 x 105 ± 0.67 x 105 copies of MOR/μg of total RNA), NMB cells (9.5 x 104 ± 4.8 x 104 copies of MOR/μg of total RNA), and U87 MG cells (3.43 x 105 ± 0.67 x 105 copies of MOR/μg of total RNA). NMB cells expressed the highest levels of DOR (3.68 x 107 ± 0.48 x 107 copies of DOR/μg of total RNA) compared to the TPA-differentiated HL-60 cells (1.94 x 106 ± 0.3 x 106 copies of DOR/μg of total RNA), undifferentiated HL-60 cells (1.26 x 106 ± 0.06 x 106 copies of DOR/μg of total RNA), SH-SY5Y cells (1.67 x 107 ± 0.41 x 107 copies of DOR/μg of total RNA), and U87 MG cells (7.86 x 105 ± 0.06 x 105 copies of DOR/μg of total RNA). TPA-differentiated HL-60 cells had the highest KOR expression (2.18 x 107 ± 0.30 x 107 copies of KOR/μg of total RNA) compared to HL-60 cells (5.55 x 106 ± 0.14 x 106 copies of DOR/μg of total RNA), NMB cells (1.17 x 107 ± 0.48 x 107 copies of DOR/μg of total RNA), SH-SY5Y cells (2.25 x 104 ± 0.32 x 104 copies of DOR/μg of total RNA), and U87 MG cells (1.21 x 105 ± 0.13 x 105 copies of DOR/μg of total RNA) (Figure 1).

Bottom Line: DOR expression was also elevated, although not significantly.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of NeuroImmune Pharmacology, Seton Hall University, 400 South Orange Ave, South Orange, NJ 07079, USA.

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.

Show MeSH
Related in: MedlinePlus