Cannabidiol (CBD) Alters the Functionality of Neutrophils (PMN). Implications in the Refractory Epilepsy Treatment

Abstract

Cannabidiol (CBD), a lipophilic cannabinoid compound without psychoactive effects, has emerged as adjuvant of anti-epileptic drugs (AEDs) in the treatment of refractory epilepsy (RE), decreasing the severity and/or frequency of seizures. CBD is considered a multitarget drug that could act throughout the canonical endocannabinoid receptors (CB1-CB2) or multiple non-canonical pathways. Despite the fact that the CBD mechanism in RE is still unknown, experiments carried out in our laboratory showed that CBD has an inhibitory role on P-glycoprotein excretory function, highly related to RE. Since CB2 is expressed mainly in the immune cells, we hypothesized that CBD treatment could alter the activity of polymorphonuclear neutrophils (PMNs) in a similar way that it does with microglia/macrophages and others circulating leukocytes. In vitro, CBD induced PMN cytoplasmatic vacuolization and proapoptotic nuclear condensation, associated with a significantly decreased viability in a concentration-dependent manner, while low CBD concentration decreased PMN viability in a time-dependent manner. At a functional level, CBD reduced the chemotaxis and oxygen consumption of PMNs related with superoxide anion production, while the singlet oxygen level was increased suggesting oxidative stress damage. These results are in line with the well-known CBD anti-inflammatory effect and support a potential immunosuppressor role on PMNs that could promote an eventual defenseless state during chronic treatment with CBD in RE.

Keywords: cannabidiol; chemotaxis; oxidative stress; oxygen consumption; polymorphonuclear neutrophils (PMNs); refractory epilepsy.

Figure 1

CBD affect the viability of PMNs. PMNs viability is significantly affected with CBD concentrations >10⁻⁵ M (A) and correlated with shorter survival time courses at the same CBD concentrations (B). Results are expressed as mean ± SD (p < 0.05). ** and *** indicate the significance differences relative to 0 M of CBD. In (B) the asterisks indicate the point from which the curves for each CBD concentration are different from the control, while the color of the asterisks indicates which CBD concentration is compared.

Figure 2

The morphology of PMNs is modified by CBD in a concentration-dependent manner. All images were captured with a magnification of 40×.

Figure 3

CBD accelerates the decrease in PMN/MN rate. (A) Spontaneous nuclear transformation from PMN to MN forms affects no more than 20% of normal PMN population in a symmetric mode. (B) A significant drop in the PMN/MN ratio is observed when PMNs are exposed to higher CBD concentrations. Results are expressed as mean ± SD (p < 0.0001). *** indicate the significance differences relative to 0 M of CBD.

Figure 4

CBD modifies the PMN chemotaxis. (A) The activation of chemotaxis by fMLP is significantly inhibited by CBD (p < 0.01). *** compared to fMLP/CBD -/- while §§§ compared to fMLP/CBD +/-. (B) During the time of the experiment, no differences were observed in the ratio PMN/MN, indicating that chemotaxis results were independent of morphologic modifications. Results are expressed as mean ± SD (p = 0.1865).

Figure 5

Oxygen uptake and chemiluminescence under several CBD concentrations. An inverse effect between oxygen uptake and chemiluminescence was observed secondary to the incubations of PMNs with increased concentrations of CBD. Results are expressed as mean ± SD (p < 0.05). * and ** indicate the significance differences relative to 0 M of CBD.