Morphine stimulates CCL2 production by human neurons

Abstract

Background: Substances of abuse, such as opiates, have a variety of immunomodulatory properties that may influence both neuroinflammatory and neurodegenerative disease processes. The chemokine CCL2, which plays a pivotal role in the recruitment of inflammatory cells in the nervous system, is one of only a few chemokines produced by neurons. We hypothesized that morphine may alter expression of CCL2 by human neurons.

Methods: Primary neuronal cell cultures and highly purified astrocyte and microglial cell cultures were prepared from human fetal brain tissue. Cell cultures were treated with morphine, and cells were examined by RNase protection assay for mRNA. Culture supernatants were assayed by ELISA for CCL2 protein. beta-funaltrexamine (beta-FNA) was used to block mu-opioid receptor (MOR)s.

Results: Morphine upregulated CCL2 mRNA and protein in neuronal cultures in a concentration- and time-dependent fashion, but had no effect on CCL2 production in astrocyte or microglial cell cultures. Immunocytochemical analysis also demonstrated CCL2 production in morphine-stimulated neuronal cultures. The stimulatory effect of morphine was abrogated by beta-FNA, indicating an MOR-mediated mechanism.

Conclusion: Morphine stimulates CCL2 production by human neurons via a MOR-related mechanism. This finding suggests another mechanism whereby opiates could affect neuroinflammatory responses.

Figure 1

Morphine effect on human neuronal chemokine production. Total RNA (5 μg) isolated from control (C) and morphine (10^-8, 10^-6 M at 24 and 48 h) exposed highly enriched neurons were used in RPA with a chemokine template. Ltn, lymphotactin; GAPDH glyceraldehydes 3-phosphate dehydrogenase.

Figure 2

Concentration-responses of morphine on human neuronal CCL2 production. Cell culture supernatants were collected from highly enriched neuronal cell cultures treated with the indicated concentrations of morphine for 24 h. Data are mean ± SEM of triplicates of three separate experiments using neurons derived from different brain specimens. *P < 0.05, **P < 0.01 versus control.

Figure 3

Effect of morphine on CCL2 production by human neurons, microglial cells, and astrocytes. Cell culture supernatants were collected from A) highly enriched neuronal cell, B) microglial cell, and C) astrocyte cultures treated with medium (control) or morphine (10^-6 M) for the given time points and assayed for CCL2 by ELISA. Data are mean ± SD of triplicates and are representative of three separate experiments using cells derived from different brain specimens. **P < 0.01 versus control.

Figure 4

Morphine effect on CCL2 production by human neurons. Immunocytochemical staining of highly purified (>95%) human neurons incubated with (A) medium and (B) morphine (10^-4 M) for 24 h was performed with (a) MAP2 antibody (neuronal marker, green), (b) CCL2 antibody (red), and (c) nuclear DAPI stain (blue). Colocalization of MAP2 and CCL2 is shown in the merged images (d).

Figure 5

Effect of MOR antagonist on morphine-mediated stimulation of neuronal CCL2 production. Highly enriched neuronal cultures were pretreated with β-FNA (3 × 10^-6 M) for 30 min prior to morphine (10^-6 M) treatment for 24 h. Supernatants were collected for CCL2 ELISA. Data are mean ± SEM of triplicates of three separate experiments using neurons derived from different brain specimens. The saturated two-way ANOVA model fit the data well with a R² of .91 and Levene's test showed no inequality of the group-wise variances (p = .36). The mean inhibitory effect of β-FNA was estimated to be 3.4 with 95% confidence interval (1.2, 5.6). **P < 0.01 versus control; ††P < 0.01 versus morphine treatment.