Β-funaltrexamine inhibits chemokine (CXCL10) expression in normal human astrocytes

Abstract

Neuroinflammation is an integral component of neurodegenerative disorders, CNS infection and trauma. Astroglial chemokines, such as CXCL10, are instrumental in neuroinflammatory signaling as well as neurotoxicity. We have utilized proinflammatory-induced CXCL10 expression in normal human astrocytes (NHA) as a model in which to assess the anti-inflammatory actions of the selective, mu-opioid receptor (MOR) antagonist, β-funaltrexamine (β-FNA). Interferon (IFN)γ+HIV-1 Tat-induced CXCL10 expression (secreted protein and mRNA) was inhibited by co-treatment with β-FNA. Neither the MOR-selective antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2 (CTAP) nor the nonselective opioid receptor antagonist, naltrexone inhibited IFNγ+HIV-1 Tat-induced CXCL10 expression. Furthermore, co-treatment with excess CTAP or naltrexone did not prevent β-FNA mediated inhibition of IFNγ+HIV-1 Tat-induced CXCL10 expression. Additionally, we utilized an inhibitor of NF-κB activation (SN50) to demonstrate that IFNγ+HIV-1 Tat-induced CXCL10 expression is NF-κB-dependent in NHA. Subsequent experiments revealed that β-FNA did not significantly affect NF-κB activation. Interestingly, we discovered that β-FNA inhibited p38 activation as indicated by decreased expression of phospho-p38. Together, these findings suggest that the inhibitory actions of β-FNA are MOR-independent and mediated, in part, via a transcriptional mechanism. These findings add to our understanding of the mechanism by which chemokine expression is inhibited by β-FNA. In conjunction with future investigations, these novel findings are expected to provide insights into the development of safe and effective treatments for neuroinflammation.

Fig. 1

β-FNA inhibits proinflammatory-induced CXCL10 expression in normal human astrocytes. Panel A) β-FNA effects on CXCL10 protein expression: In the presence or absence or β-FNA (10 μM), normal human astrocytes were exposed to human recombinant IFNγ (10 ng/ml), HIV-1 Tat_1–72 (100 nM), IFNγ + HIV-1 Tat_1–72, or serum free medium alone for 24 h. CXCL10 protein levels in the media were then measured by ELISA. The data represent mean + S.E.M of 9 independent experiments. Two-way ANOVA (β-FNA treatment × stimulus) indicated a significant effect of β-FNA (p < 0.009), stimulus (p < 0.0001) and an interaction (p < 0.0005). Bonferroni’s multiple comparisons indicated that β-FNA significantly (* p < 0.001) inhibited IFNγ + HIV-1 Tat_1–72-induced CXCL10 expression. Panel B) β-FNA effects on CXCL10 mRNA expression: In the presence or absence or β-FNA (10 μM), normal human astrocytes were exposed to human recombinant IFNγ (10 ng/ml) + HIV-1 Tat_1–72 (100 nM) or in serum free medium alone for 8 h. Total RNA was isolated and CXCL10 and GAPDH mRNA assessed by real-time PCR. The data represent mean + S.E.M of 6 independent experiments. Analysis of ΔC_T values by one-way ANOVA followed by Newman-Keuls multiple comparisons indicated that β-FNA significantly inhibited IFNγ + HIV-1 Tat_1–72-induced CXCL10 mRNA expression. * p < 0.05 vs. IFNγ + HIV-1 Tat_1–72

Fig. 2

β-FNA inhibition of IFNγ + HIV-1 Tat_1–72-induced CXCL10 protein expression in normal human astrocytes is not affected by co-exposure to either naltrexone (Panel A) or CTAP (Panel B). Normal human astrocytes were exposed to human recombinant IFNγ (10 ng/ml) + HIV-1 Tat_1–72 (100 nM) alone or in the presence of β-FNA (10 μM), naltrexone or CTAP (30 and 100 μM), or β-FNA + naltrexone or CTAP for 24 h. CXCL10 protein levels in the media were then measured by ELISA. The data are expressed as % control (relative to IFNγ + HIV-1 Tat_1–72) and represent mean + S.E.M of 2–4 independent experiments; triplicate measures (wells) in each experiment. Data were analyzed using one-way ANOVA and Dunnett’s post test comparison. * p < 0.05 vs. IFNγ + HIV-1 Tat_1–72

Fig. 3

IFNγ + HIV-1 Tat_1–72-induced CXCL10 protein expression in normal human astrocytes is NF-κB- dependent. CXCL10 expression was induced in normal human astrocytes by stimulating with human recombinant IFNγ (10 ng/ml) + HIV-1 Tat_1–72 (100 nM) for 24 h. To assess the role of NF-κB, the inhibitor of NF-κB nuclear translocation, 50 μM SN50 (or 50 μM of the inactive control peptide, SN50 Mut), was added to cell cultures 1 h prior to stimulation. The data represent mean + S.E.M of 2 independent experiments; triplicate measures (wells) in each experiment. Data were analyzed using one-way ANOVA and Newman-Keuls post test comparison. * p < 0.05 vs. IFNγ + HIV-1 Tat_1–72

Fig. 4

β-FNA does not inhibit IFNγ + HIV-1 Tat_1–72, -induced NF-κB p65-DNA binding activity in normal human astrocytes. In the presence or absence or β-FNA (10 μM), normal human astrocytes were exposed to human recombinant IFNγ (10 ng/ml) + HIV-1 Tat_1–72 for 10–270 min. NF-κB activation was indicated by increased levels of the active (DNA-binding) form of NF-κB p65 in the nucleus. The data represent mean + S.E.M of 3 independent experiments. Two-way ANOVA (β-FNA treatment × time) indicated a significant effect of time (p < 0.004); whereas, there was neither a significant effect of β-FNA (p = 0.59), nor an interaction (p = 0.62). * p < 0.05 vs. 10 min as indicated by Bonferroni’s multiple comparisons. Binding activity in unstimulated cells was 43 ± 7 arbitrary units/μg protein.

Fig. 5

β-FNA inhibits IFNγ + HIV-1 Tat_1–72, -induced p38 activation in normal human astrocytes. In the presence or absence or β-FNA (10 μM), normal human astrocytes were exposed to human recombinant IFNγ (10 ng/ml) + HIV-1 Tat_1–72 for 10–270 min. Panel A): Western blot was used to determine levels of total p38 and phosphorylated-p38 (p-p38) in the cytosolic fraction. Panel B): Data for the densitometric analysis represent mean + S.E.M of 3–5 independent experiments. Integrated density values are presented as a ratio of p-p38/total p38 and expressed as fold change relative to unstimulated control.. Data were analyzed using one-way ANOVA and Newman-Keuls post test comparison. ** p < 0.01 vs. IFN γ + HIV- 1 Tat_1–72 at 10 min; *** p < 0.001 vs. IFNγ + HIV-1 Tat_1–72 at 30 min.