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

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Description

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 10 5 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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Publié le 01 janvier 2012
Nombre de lectures 12
Langue English
Poids de l'ouvrage 1 Mo

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Byrne et al. Journal of Neuroinflammation 2012, 9:252 JOURNAL OF
http://www.jneuroinflammation.com/content/9/1/252 NEUROINFLAMMATION
RESEARCH Open Access
Interleukin-1 beta-induced up-regulation of
opioid receptors in the untreated and
morphine-desensitized U87 MG human
astrocytoma cells
1 1,3† 1,2† 1,2*Linda Staikos Byrne , Jinsong Peng , Sraboni Sarkar and Sulie L Chang
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
5the MOR, DOR and KOR, U87 MG cells (2 x 10 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.
Keywords: IL-1β, Morphine, Mu-opioid receptor, U87 MG astrocytoma cells
* Correspondence: sulie.chang@shu.edu

Equal contributors
1
Institute of NeuroImmune Pharmacology, Seton Hall University, 400 South
Orange Ave, South Orange, NJ 07079, USA
2
Department of Biological Sciences, Seton Hall University, 400 South Orange
Ave, South Orange, NJ 07079, USA
Full list of author information is available at the end of the article
© 2012 Byrne et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.Byrne et al. Journal of Neuroinflammation 2012, 9:252 Page 2 of 12
http://www.jneuroinflammation.com/content/9/1/252
Background IL, USA). Interleukin-1 beta (IL-1β)andinterleukin-1re-
In the brain, astrocytes are an important component of ceptor antagonist protein (IL-1RAP) were obtained from
the blood–brain barrier and participate in the mainten- R&D Systems (Minneapolis, MN, USA).
ance of homeostasis. They are also a main producer of
cytokines and chemokines [1]. In addition, astrocytes are U87 MG cells
capable of surviving under inflammatory conditions and Human astrocytoma cells (U87 MG), which have been
are resistant to death receptor-mediated apoptosis [2]. previously used to represent astrocytes in in vitro studies
Interleukin-1 (IL-1) is a pro-inflammatory cytokine [18-21], were obtained from American Type Culture
expressed in the central nervous system (CNS). It is pro- Collection (ATCC) (Rockville, MD, USA) and grown in
duced by a wide variety of cells, such as glia, astrocytes, DMEM containing 10% FBS and 1% penicillin/strepto-
neurons, monocytes and endothelial cells [3,4]. During mycin sulfate in a humidified 5% CO at 37°C.2
inflammation, IL-1 can activate the paraventricular nu-
cleus of the hypothalamus, resulting in the release of HL-60 cells
corticotrophin-releasing hormone and subsequent activa- Human promyelocytic leukemia (HL-60) cells were
tion ofthe hypothalamic-pituitary-adrenal(HPA) axis[4]. obtained from ATCC (Rockville, MD, USA) and cultured
Three opioid receptors have been identified to date – in RPMI-1640 medium supplemented with 20% FBS and
mu (MOR), delta (DOR) and kappa (KOR). Morphine, 1% penicillin/streptomycin sulfate. Cells were main-
which has a higher affinity for the MOR compared to tained at 37°C in humidified 5% CO . The HL-60 cells2
the KOR and DOR [5-7], elicits its effects mainly were induced to differentiate into macrophage/mono-
through the MOR. Morphine is well known for its anal- cyte-like cells with TPA (16 nM TPA/0.1% EtOH in
gesic effects and addictive properties [8], as well as its RPMI-1640 medium) for 4 d. The TPA-treated medium
ability to alter the endocrine and immune systems was changed every 48 h until the completion of
[9,10]. Chronic morphine use can cause immunosup- differentiation.
pression, ande addicts often have an increased
incidence of viral hepatitis, bacterial pneumonias, endo- NMB cells
carditis, tuberculosis and CNS infections [11,12]. Con- Human neuroblastoma cells (NMB cells) were a gift
versely, chronic morphine exposure can also indirectly from Dr. Horace H. Loh (University of Minnesota,
potentiate an immune response by desensitizing the HPA MN, USA). NMB cells were grown and maintained in
axis [9,13,14], and increasing the production and activity RPMI-1640 medium containing 10% FBS. NMB cells
of various cytokines, including IL-1β [15], IL-6 [16], and were maintained in a humidified environment of 5%
TNF-α [14]. Early studies showed that IL-1 increases the CO at 37°C.2
expression of opioid receptors in primary human glial
cells [17] and human brain microvascular primary cells SH-SY5Y cells
[3], suggesting that this pro-inflammatory cytokine may Human neuroblastoma cells (SH-SY5Y cells) were a gift
possess neuromodulatory effects and may be an import- from Dr. Robert Ross (Fordham University, New York,
ant component in the immune-opioid circuit. NY, USA). SH-SY5Y cells were grown and maintained
In this study, we hypothesized that a functional relation- in a 1:1 mixture of Earle's Minimum Essential Medi-
ship may exist between IL-1β and the opioid receptors in um, Ham's Nutrient Mixture F12 and 10% FBS with
human astrocytes. We examined the ability of IL-1β to in- penicillin/streptomycin sulfate.
crease expressionof the MOR,DOR andKORinahuman
astrocytic cell line, U87 MG, both in the untreated state as IL-1β treatment
5
well as in the state desensitized with morphine. We then U87 MG cells (2 x 10 cells/well) were treated with cell
examined whether IL-1β-induced MOR up-regulation is culture medium containing either vehicle (cell culture
mediatedthrough the IL-1β receptor. medium) or IL-1β (20 ng/mL or 40 ng/mL) for 0, 3, 6,
12, 24, or 48 h. The medium was aspirated and 1 mL
WMethods TRIzol was added to each well. The cells were then
Materials frozen and stored at −80°C for further analysis.
W
Cell culture and TRIzol reagents were obtained from
GIBCO/Invitrogen (Carlsbad, CA, USA). Morphine, na- Co-treatment with IL-1β and IL-1RAP
5
loxone and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) U87 MG cells (2 x 10 cells/well) were treated with cell
and all other reagents used for RNA extraction were culture medium containing either vehicle (cell culture
obtained from Sigma (St. Louis, MO, USA). DAPI was medium), IL-1β (20 ng/mL), IL-1RAP (400 ng/mL)+
obtained from Pierce (Rockford, IL, USA), and antibodies vehicle, IL-1RAP (400 ng/mL)+IL-1β (20 ng/mL),
to the MOR were obtained from Chemicon (Rosemont, IL-1RAP (4,000 ng/mL)+vehicle, or IL-1RAP (4,000 ng/mL)Byrne et al. Journal of Neuroinflammation 2012, 9:252 Page 3 of 12
http://www.jneuroinflammation.com/content/9/1/252
+IL-1β (20 ng/mL). IL-1RAP concentrations (400 ng/mL 37°C. The medium was removed and the cells were washed
and 4,000 ng/mL) exceeded the manufacturer’s recom- twice with 1X PBS, then lysed with 0.1 N HCl. The cell
mendation of a 1:100 ratio of IL-1β to IL-1RAP needed lysates were frozen at−20°C until intracellular cAMP levels
for IL-1RAP to be effective. Cells were then incubated in were measured using a commercially available RIA
5% CO at 37°C for 12 h. The medium was aspirated and kit (Amersham Biosciences, Inc., Piscataway, NJ, USA). Par-2
W
1mLTRIzol was added toeach well. The cells were then allel studies were conducted using two opioid peptides with
frozenand storedat−80°Cfor further analysis. a high affinity for the MOR, endomorphin-1 (10 μM) or
endomorphin-2 (10μM), in place of morphine.
Time course of morphine’s effects on the MOR
5
U87 MG cells (1.5 x 10 cells/well) were treated with fresh Radioimmunoassay (RI

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