Cannabis (Cannabis sativa
L., fam. Cannabaceae) is accredited with several medicinal properties [1
], and conclusive evidence gives support to its therapeutic benefits in the treatment of chronic pain, multiple sclerosis, as well as chemotherapy-induced nausea and vomiting [2
]. Currently, medical cannabis is available in many countries, however, a world of controversy still surrounds cannabis use in clinical practice in consideration of its mind-altering properties and addictive potential related to its ∆9
-tetrahydrocannabinol (THC) content [3
]. As a result, new varieties of cannabis have been developed which are poor in THC and rather rich in non-psychoactive cannabinoids [4
]. Cannabidiol (CBD, Figure 1
) is the major non-psychoactive cannabinoid and occurs naturally in appreciable amounts in the leaves, and flowers of cannabis plants [5
]. CBD is devoid of any drug abuse liability [6
] and carries no meaningful side effects across a wide dose range in humans (up to 6000 mg/day p.o.) [7
]. CBD has recently received Food and Drug Administration (FDA) approval for seizures associated with Lennox-Gastaut syndrome or Dravet syndrome [10
], furthermore available evidence suggests that CBD might be beneficial in a number of diseases, including: Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, epilepsy, Huntington’s disease, hypoxia-ischemia injury, pain, anxiety, depression, cancer, nausea, inflammatory diseases, infections, rheumatoid arthritis, inflammatory bowel and Crohn’s diseases, cardiovascular disease and diabetic complications [11
]. Preliminary studies suggest that most of the therapeutic effects of cannabis and CBD may be linked to their ability to affect immunity and inflammation [12
], nonetheless the underlying mechanisms are not yet plainly understood.
Polymorphonuclear leukocytes (PMN) play a critical role in the inflammatory process as the first-line of defence against invading microorganisms [13
]. PMN are sensitive to agents such as bacterial by-products as well as to cytokines and chemokines [14
]. Thereafter, migrating into the site of inflammation, they produce and release proinflammatory and antimicrobial mediators, including cytokines and chemokines, as well as oxidative metabolites such as reactive oxygen species (ROS). Rapid resolution of inflammation usually leads to tissue repair, however chronicization of the process results in tissue damage, which is considered pivotal in a wide range of inflammatory diseases [15
]. PMN are therefore attractive targets for the development of novel anti-inflammatory therapeutics in many different clinical settings [16
Circumstantial evidence suggests that inhibition of PMN functions could be a potential mechanism underlying the anti-inflammatory effects of CBD. Indeed, in mice, it has been reported that CBD suppresses PMN polarization towards the proinflammatory N1 phenotype [18
], attenuates myeloperoxidase activity [19
], and inhibits respiratory burst [20
]. Evidence in human PMN includes so far inhibition of oxidative metabolism [20
], adhesion to the activated endothelium [21
] as well as cell migration [20
]. On the other hand, however, there is no evidence regarding the activity of cannabis on PMN. Therefore, the present study was conducted to examine the effects of CM5, a Cannabis sativa
L. extract standardized in 5% CBD and with a low content of THC (<0.2%), on human PMN functions, including cell migration, oxidative metabolism and production of tumour necrosis factor (TNF)-α. We then systematically compared CM5 with pure CBD, in order to determine whether the effects of cannabis on PMN could be ascribed to its content in CBD.
The present study provides evidence that CM5, a standardized cannabis extract in CBD 5% and with THC <0.2%, extensively affects human PMN functions. Specifically, at non-cytotoxic concentrations, CM5 was found to effectively inhibit stimulated migration, oxidative metabolism and production of the proinflammatory cytokine TNF-α. Comparison with the effects of pure CBD suggests that most of the activity of CM5 could be explained by its CBD content. However, CM5 at equimolar concentration of CBD was more potent and effective than CBD alone on PMN migration, as well as on TNF-α production. Moreover, CBD alone was more effective than CM5 on PMN oxidative metabolism.
To our best knowledge, this is the first study reporting the inhibitory effects of a cannabis extract rich in CBD and with a low level of THC on human PMN functions. With respect to CBD, however, our findings support previous research showing the ability of CBD to inhibit the oxidative metabolism [20
] and to attenuate PMN migration [22
], and extend for the first time the observations to TNF-α production. Importantly, the inhibitory effects of CBD occurred at concentrations achieved with doses used clinically for the treatment of drug-resistant seizures in Lennox–Gastaut syndrome or Dravet syndrome (up to 20 mg per kg of body weight per day) [23
], suggesting that our findings can be easily translated into clinics.
CM5 is a complex mixture containing 5% of CBD and 95% of cannabis vegetal complex matrix (including compounds such as other cannabinoids, flavonoids, fatty acids). Our study revealed that CBD alone reproduces the inhibitory activity of CM5 on human PMN functions, suggesting that the effects of CM5 on PMN could be ascribed chiefly to its CBD content. However, we observed subtle differences in terms of potency and efficacy between CM5 and CBD, indicating that beyond CBD, other components occurring in CM5 may contribute to its inhibitory effects on PMN. Thus, the higher activity of CM5 on PMN migration and TNF-α production may result from synergistic and/or additive interactions between its various components. On the other hand, the lower activity of CM5 on oxidative burst may stem from antagonistic interactions. Further research is, therefore, needed for the identification, isolation as well as pharmacological characterization of components present in cannabis and effectively contributing to its overall activity on PMN.
Deciphering the mechanism underlying the inhibitory effects of CM5 and CBD on PMN functions was beyond the scope of our study. However, clues from prior research suggest that CBD may exert its effects on PMN possibly through CB2
, at least in rodent models [24
], but also by acting on other molecular targets distinct from CB1
]. Future mechanistic studies to determine the molecular basis of the inhibitory effects of CBD on PMN should focus on CBD targets that are expressed on human PMN such as CB2
], GPR55 [26
], PPARγ [27
] and TRPV1 [28
PMN are recruited to sites of inflammation where they eradicate pathogens through various effector mechanisms including degranulation, phagocytosis, generation of ROS as well as neutrophil extracellular traps (NETs) formation [29
]. However, excessive PMN responses may contribute to ongoing inflammation and tissue damage in a number of diseases and ailments [32
]. Compelling evidence supports the use of cannabis and CBD in a plethora of pathological conditions, some of which stem from the exacerbation of the inflammatory response of PMN and include pain [36
], multiple sclerosis [37
], diabetic complications [38
], inflammatory bowel disease [39
], cardiovascular diseases [40
], hypoxia-ischemia injury [41
], rheumatoid arthritis [35
], cancer [42
], and Crohn’s disease [43
]. Collectively, our findings point to PMN as potential targets for the therapeutic actions of cannabis and CBD.
Since PMN activation results in a much more complex pattern of functional changes that, in addition to activation of migration, respiratory burst and cytokine production, also include phagocytosis, production and release of proteolytic enzymes and NETs as well as interaction with other cells, a great deal of knowledge is still required before a firm conclusion can be drawn about the inhibitory effects of CM5 and CBD on PMN. Moreover, as the effects on TNF-α were not so intense as those on migration and oxidative metabolism, any difference in receptor and/or signal transduction pathways involved might deserve consideration. In any case, available evidence is encouraging and now strongly warrants careful in-depth characterization in order to provide a rational basis for better exploiting the potential health benefits of cannabis or its derivative in broad-impact pathological conditions including pain, diabetic complications, cancer and cardiovascular diseases.