Naltrexone Inhibits IL-6 and TNFα Production in Human Immune Cell Subsets following Stimulation with Ligands for Intracellular Toll-Like Receptors

Abstract

The opioid antagonist naltrexone hydrochloride has been suggested to be a potential therapy at low dosage for multiple inflammatory conditions and cancers. Little is known about the immune-modulating effects of naltrexone, but an effect on the activity of toll-like receptor 4 (TLR4) has been reported. We analyzed the effects of naltrexone hydrochloride on IL-6 secretion by peripheral blood mononuclear cells (PBMC) in vitro following stimulation with ligands for TLR4 and for the intracellular receptors TLR7, TLR8, and TLR9. Naltrexone did not affect cell viability or induce apoptosis of PBMC. Intracellular staining demonstrated that naltrexone inhibited production of IL-6 and TNFα by monocyte and plasmacytoid dendritic cell subsets within the PBMC population following treatment with ligands for TLR7/8 and TLR9, respectively. No effect of cytokine production by PBMC following stimulation of TLR4 was observed. Additionally, naltrexone inhibited IL-6 production in isolated monocytes and B cells after TLR7/8 and TLR9 stimulation, respectively, but no effect on IL-6 production in isolated monocytes after TLR4 stimulation was observed. These findings indicate that naltrexone has the potential to modulate the secretion of inflammatory cytokines in response to intracellular TLR activity, supporting the hypothesis that it may have potential for use as an immunomodulator.

Keywords: B cells; interleukin-6; monocytes; naltrexone; plasmacytoid dendritic cells; toll-like receptor; tumor necrosis factor alpha.

Figures

Figure 1

Naltrexone inhibits IL-6 production after toll-like receptor (TLR)7/8 and TLR9 stimulation but not after TLR4 or IL-1 stimulation. 1 × 106 peripheral blood mononuclear cells were incubated with (A) 1 ng/ml LPS (TLR4-L), (B) 1µM R848 (TLR7/8-L), (C) 1µM CpG (TLR9-L), (D) 100 ng/ml IL-1 (IL-1R) in the presence or absence of 1–200µM naltrexone for 24 h. Cell-free supernatants were collected and analyzed for IL-6 by ELISA. Data show the mean; SD values are shown and were analyzed using an one-way ANOVA and Tukey’s multiple comparison test (n = 5 TLR ligand experiments and n = 3 IL-1). *p < 0.05.

Figure 2

Intracellular cytokine staining for TNFα and IL-6 in monocytes and plasmacytoid dendritic cells. 1 × 106 peripheral blood mononuclear cells (PBMC) were incubated with either LPS 1 ng/ml (A,C), R848 1µM (B), or CpG 1µM (D) and 200 µM naltrexone for 6 h in the presence of brefeldin A for 4 of those hours. After 6 h, PBMC were stained using antibody panel shown in Figure S4 in Supplementary Material and stained for either intracellular IL-6 or TNF-α. Results show the mean fluorescence intensity (MFI) of IL-6 or TNF-α within that subset from 5 donors. Histograms are representative of 5 independent experiments.

Figure 3

Naltrexone inhibits IL-6 production in isolated monocytes and B cells after toll-like receptor (TLR)7/8 and TLR9 stimulation, respectively, but has no effect on IL-6 production in isolated monocytes after TLR4 stimulation. (A) CD14+ monocytes were isolated from peripheral blood mononuclear cells (PBMC) using magnetic bead isolation. 1 × 105 CD14+ cells were incubated with 1 ng/ml LPS (TLR4-L) or 1µM R848 (TLR7/8-L), in the presence or absence of 200µM naltrexone for 24 h. Cell-free supernatants were collected and analyzed for IL-6 by ELISA. (B) CD19+ B cells were isolated from PBMC using magnetic bead isolation. 105 B cells were incubated with 1µM CpG or 3 µg/ml CD40-L and 20 ng/ml IL-4, with or without 200µM naltrexone for 24 h. IL-6 production was measured in cell-free supernatants by ELISA. Data show the mean and SD values (n = 4).

Figure 4

Toll-like receptor ligand (TLR-L) and naltrexone does not affect the viability of peripheral blood mononuclear cells (PBMC). (A) 1 × 106 PBMC were incubated with 1–200µM naltrexone for 24 h before percentage viability was assessed using trypan blue exclusion. (B,C) 1 × 106 PBMC were incubated with 1 ng/ml LPS (TLR4-L), 1µM R848 (TLR7/8-L), 1µM CpG (TLR9-L), and 200µM naltrexone for 24 h. PBMC were incubated with annexin V and 7AAD before being analyzed by flow cytometry. Figure 4B shows the gating strategy, and Figure 4C shows results from 4 donors. AV−7AAD− are viable cells, AV+7AAD− are in early apoptosis, and AV+7AAD+ are in late apoptosis.