Now is not the time for partisan arguments or preserving the status quo of powerful and influential industries. The Profile of Immune Modulation by Cannabidiol (CBD) Involves Deregulation of Nuclear Factor of Activated T Cells (NFAT) Corresponding author: Norbert E. Kaminski, Center for Integrative Immune Responses Regulated by Cannabidiol Department of Basic Sciences, Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State,
How nuclear power and hemp can help solve scarcity
Now is not the time for partisan arguments or preserving the status quo of powerful and influential industries.
The last two years have been a wakeup call for humanity: Decades of dire warnings from experts in nearly every scientific discipline have converged on us seemingly all at once. We are mired in a global pandemic that has exposed weaknesses in — and created major interruptions to — the supply chains for food, fuel, labor, and goods. We’ve experienced significant weather events and shifts in seasonal weather behavior that can only be attributed to the human impact on climate change. Species are going extinct at an alarming rate. Plastic pollution is choking our oceans and land. The list just goes on and on.
Scarcity is the root of all our major problems
For generations, most of the world’s problems have been attributed to the scarcity of resources — who has it, who doesn’t, and what means will be undertaken to control it or obtain it. Wars, inequality, hunger, and economic turmoil ultimately boil down to the haves and the have nots on a global scale.
Now, likely as an outcome of this overall scarcity, a Central European conflict instigated by Russia upon the nation of Ukraine is leading towards massive increases in global petroleum and natural gas prices, in addition to causing potential interruption of about 25% of the world’s grain supply that is locked up in Russia and Ukraine.
As just one opinionated technologist, I won’t presume to have solutions for all of the world’s extremely complicated economic and geopolitical problems. But I believe that individual nations can take specific actions to minimize these impacts on their own citizens.
What to do about the fuel
Let’s start with petroleum and natural gas — two resources that are not renewable, in limited supply, and controlled by a limited number of nations.
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In addition to being the world’s largest source of fuel, petroleum is used to make products such as plastics, polyurethane, solvents, and hundreds of other applications. We also need to find ways to replace it in the petrochemical industry.
Even assuming we can eventually switch many vehicles to EV-based propulsion, there is also the issue of the aviation, marine, and commercial transportation industries needing a viable fuel source.
Hemp, which was legalized for cultivation with restrictions in 2018 as part of the Farm Bill, is an extremely good candidate for doing this because, as a crop, it has many different applications.
Hemp cultivation does not require chemicals or irrigation. It proliferates with a very high yield — it takes about four months for a stalk to reach maturity, and it is possible with efficient farming techniques to produce 500 to 1500 pounds of hemp biomass per acre.
Overall, experts have concluded that “meeting US demands for oil and gas would require intensive cultivation of only about 6% of the land area of the 48 contiguous states, or just over 116 million acres”, according to the Great Book of Hemp by Rowan Robinson
Hemp fibers can be used not only for biofuel but also for textile and hundreds of other manufacturing uses, including biodegradable hemp plastic.
Hemp produces very high-quality cooking oil, and its seeds are very high in protein, making it ideal for food industry applications such as hemp milk, hemp oil, hemp cheese substitutes, and hemp-based protein powder — all of which we will need as plant-based proteins become more and more incorporated into our diets. According to the Agricultural Marketing Resource Center, one acre of hemp can yield an average of 700 pounds of grain which can be pressed into about 22 gallons of oil and 530 pounds of meal.
Which hemp cultivars we would use for these purposes is really up to the scientists and the agriculture industry — but some of the most promising candidates in terms of oil yield are likely to be currently classified as marijuana due to their THC content, as from a legal perspective, anything over 0.3% THC on a dry weight basis is no longer considered to be industrial hemp.
I have long been a proponent of the federal decriminalization or legalization of marijuana for its many therapeutic and pharmacological properties, which have been investigated academically in other nations, such as Israel, a world leader in this area. Initial pre-peer review studies by the University of Chicago under grants from the National Institute of Health suggest that CBD is a potent anti-inflammatory agent and antiviral treatment.
Assuming these observations are conclusive, CBD and other cannabinoids have the potential to be the best treatment yet for COVID-19 and other viral diseases, among other debilitating ailments, which along with existing and future vaccines, would help combat the current labor (and ultimately, supply chain) problems associated with the pandemic by reducing the duration of illness or preventing or even halting serious illness entirely.
While we would need to likely spend hundreds of millions or even billions of dollars of research on all of the potential pharmaceutical applications, as marijuana contains hundreds of compounds for investigation, our first step towards energy independence may very well be the wholesale legalization of marijuana, not just expanding hemp production from the original Farm Bill.
At the very least, a large-scale hemp industry would employ millions of people across multiple sectors, from food and industrial applications to fuel and pharmaceuticals.
What to do about power generation
But biofuels address only part of the overall energy independence challenge. The United States’ power-generating infrastructure is largely based on non-renewable energy.
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According to the US Energy Information Administration (EIA), in 2021, the United States generated about 4 trillion kilowatt-hours of electricity. Approximately 60% of the electricity generated was from fossil fuels (coal, natural gas), with 19% attributed to nuclear and 21% to renewables.
A report released by the US EIA in March of 2022 forecasts an upward trend towards the use of renewables as 44% of the country’s total power generation capacity by 2050.
However, with the future of Russian oil and natural gas exports to the West now in question, the EIA’s model will require major recalculations. In particular, how will this supply shortfall impact our previous calculations from a scarcity perspective? But complex economic models aren’t needed for us to conclude that if we don’t make some changes to how we generate power, the future of the climate and global economy looks less than promising.
We will need to look at renewables such as wind, solar, hydroelectric, and geothermal. To accommodate the renewables, nations like the United States will need to build two-way electrical grids, not just the traditional Transmission System Operator and Distribution System Operator model we have been using for more than 100 years. Modernized, distributed smart grids — built on open source technologies — will need to be implemented, such as the ones France’s RTE and the Netherlands’ Alliander are building.
Renewables are essential. But we need to face that 800-pound gorilla. While we can supplement the world’s energy requirements with renewables, it is highly unrealistic to think we can supplant coal and natural gas with them. Any discussion of eliminating the fundamental fuel sources for large-scale power generation on a global basis needs to include nuclear.
In 2010, I postulated that Barack Obama could have been the Nuclear President. Twelve years later, my position is unchanged. According to the US Energy Information Administration, there are 93 nuclear reactors distributed among 56 atomic power plants in 28 states, generating approximately 800,000 Mwh of energy. As stated earlier, that represents about 19% of the energy consumed in the United States.
Back of the envelope calculations, based on current monthly nuclear energy output figures tracked by the EIA and 4 trillion kilowatt-hour consumption estimates indicate that we would need between 200 or 300 or so new reactors to replace all fossil fuel electricity in the United States, and perhaps as many as 860 new reactors to replace fossil fuels for all domestic uses, including transportation and heating/cooling.
This assumption is based on the existing power generation infrastructure being fungible and on current power output per reactor (many of which are 50-year-old designs), without modern efficiencies of innovation being applied such as breeder capability and modern construction techniques which may permit smaller (and much safer) designs.
While total replacement of the coal and natural gas power generating infrastructure entirely with nuclear may be unrealistic, if we do these calculations and build models with increased use of renewables and a modernized grid into consideration, it may be possible to achieve our energy goals with considerably fewer new nuclear plants.
The safety discussion needs to be had, but I think at this point, we are well beyond NIMBY-ism as a divisive element to hold back new nuclear reactor buildouts. The modern designs used by the US Navy and tested by the government at DARPA and other agencies are virtually meltdown proof and have many new safety enhancements that make them extremely safe to operate.
The arguments from those who protest nuclear energy’s use have been debunked, many times. Would we need to heavily safeguard these plants from the potential enemy military, terrorist, and cyberattacks? Yes. But we need to do this for all of our infrastructures anyway. Is sustainable, commercial nuclear fusion a possibility in the next 30 years? Recent developments indicate that it could happen. But we need nuclear fission as a bridge technology to get us there.
Ending the status quo
Now is not the time for partisan arguments or preserving the status quo of powerful and influential industries about how we approach the situation. We are now at an inflection point where the country’s economic wellbeing and national security are at stake.
We’ve seen in decades past critical strategic decisions made and accelerated paradigm shifts in technology occur due to pressing needs, such as in wartime. With the world in crisis and energy scarcity being the most pressing topic affecting virtually everything, we again need to make some important choices about how we proceed.
The first step is to realize that the status quo is no longer an option. We need to rapidly move away from fossil fuels and towards renewable energy sources. The time for partisanship is over; we need to come together and make this shift happen.
We have the technology available to make this happen, but it will require a concerted effort from all sectors of society. The government needs to provide the necessary regulation and incentives to make it happen, while the private sector needs to invest in the research and development of new technologies. And everyday citizens need to do their part by making choices that help reduce our reliance on fossil fuels.
The Profile of Immune Modulation by Cannabidiol (CBD) Involves Deregulation of Nuclear Factor of Activated T Cells (NFAT)
Corresponding author: Norbert E. Kaminski, Center for Integrative Toxicology, Michigan State University, 315 National Food Safety and Toxicology Building, East Lansing, MI 48824; Telephone, 517-353-3786; Fax, 517-432-3218; e-mail, [email protected]
Cannabidiol (CBD) is a cannabinoid compound derived from Cannabis Sativa that does not possess high affinity for either the CB1 or CB2 cannabinoid receptors. Similar to other cannabinoids, we demonstrated previously that CBD suppressed interleukin-2 (IL-2) production from phorbol ester plus calcium ionophore (PMA/Io)-activated murine splenocytes. Thus, the focus of the present studies was to further characterize the effect of CBD on immune function. CBD also suppressed IL-2 and interferon-γ (IFN-γ) mRNA expression, proliferation, and cell surface expression of the IL-2 receptor alpha chain, CD25. While all of these observations support the fact that CBD suppresses T cell function, we now demonstrate that CBD suppressed IL-2 and IFN-γ production in purified splenic T cells. CBD also suppressed activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) transcriptional activity, which are critical regulators of IL-2 and IFN-γ. Furthermore, CBD suppressed the T cell-dependent anti-sheep red blood cell immunoglobulin M antibody forming cell (anti-sRBC IgM AFC) response. Finally, using splenocytes derived from CB1 -/- /CB2 -/- mice, it was determined that suppression of IL-2 and IFN-γ and suppression of the in vitro anti-sRBC IgM AFC response occurred independently of both CB1 and CB2. However, the magnitude of the immune response to sRBC was significantly depressed in CB1 -/- /CB2 -/- mice. Taken together, these data suggest that CBD suppresses T cell function and that CB1 and/or CB2 play a critical role in the magnitude of the in vitro anti-sRBC IgM AFC response.
Cannabinoids are a group of structurally-related compounds derived from the Cannabis Sativa plant, which is commonly known as marijuana. The primary psychoactive congener in marijuana is tetrahydrocannabinol (THC) . Although THC is currently approved for medical use as Marinol ® , there exists an ongoing debate in the United States as to whether smoking crude marijuana could be a medical necessity. This debate has sparked interest in determining the physiological properties of some of the other plant-derived cannabinoid compounds. One such compound is cannabidiol (CBD), which is one of the most abundant cannabinoids in the plant.
CBD possesses low affinity for both CB1 and CB2 cannabinoid receptors and therefore, does not produce the “high” associated with marijuana use [2, 3]. Despite this, CBD does exhibit immunosuppressive properties. In particular, CBD decreased IL-8 and the chemokines MIP-1α and MIP-1β from a human B cell line . CBD has also been shown to suppress collagen-induced arthritis , and carrageenan-induced inflammation . Importantly, CBD has been efficacious in combination with THC in treating neuropathic pain in multiple sclerosis, an autoimmune disease [7, 8].
Despite these reports that CBD possesses immunosuppressive actions, its effects on T lymphocytes have not been fully characterized. With our previous demonstration that CBD was one of the more potent plant-derived cannabinoids in suppressing IL-2 from PMA/Io-stimulated splenocytes , the focus of the present studies was to further investigate the effects of CBD on T lymphocyte function. The immunological endpoints include the determination of the effect of CBD on cytokine production (IL-2 and IFN-γ) from splenocytes activated through the T cell receptor, T and B cell proliferation, AFC responses, and direct effects on purified splenic T cells. As many reports in the literature suggest the involvement of a yet unidentified putative third cannabinoid receptor [10, 11], cannabinoid actions via other receptors [12-14], and that some effects of CBD can be reversed by the CB1 and CB2 receptor antagonists , we utilized splenocytes derived from CB1 -/- /CB2 -/- mice to address the role of CB1 and CB2 in the effects of CBD in T lymphocytes. Our results suggest that CBD suppresses T cell function via a mechanism that involves AP-1 and NFAT, and we have also discovered a putative critical role for CB1 and/or CB2 in the magnitude of the in vitro anti-sRBC IgM AFC response.
2. Materials and methods
CBD and THC were provided by the National Institute on Drug Abuse (Bethesda, MD). All other reagents were obtained from Sigma (St. Louis, MO) unless otherwise noted.
Pathogen-free female B6C3F1 or C57BL/6 mice, 6 weeks of age, were purchased from Charles River Breeding Laboratories (Portage, MI). On arrival, mice were randomized, transferred to plastic cages containing sawdust bedding (5 animals/cage), and quarantined for 1 week. CB1 -/- /CB2 -/- mice were kindly provided by Dr. Andreas Zimmer (University of Bonn) and were bred at Michigan State University. Mice were given food (Purina Certified Laboratory Chow) and water ad libitum and were not used for experimentation until their body weight was 17-20 g. Animal holding rooms were kept at 21-24°C and 40-60% relative humidity with a 12-hr light/dark cycle. All procedures involving mice were performed in accordance with guidelines set forth by the Institutional Animal Care and Use Committee at Michigan State University.
2.3 Preparation of lymphocyte cultures
Mice were sacrificed and spleens were aseptically removed. Single cell suspensions were prepared and cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 5 × 10 -5 M 2-mercaptoethanol, and 2-10% bovine calf serum (BCS; Hyclone, Logan, UT). For immunofluorescence analysis, erythrocytes were lysed with ACK solution (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). Jurkat cells (clone E6-1, ATCC, Manassas, VA) were maintained in RPMI 1640 medium supplemented with 2-10% BCS, 100 units/ml penicillin, 100 μg/ml streptomycin, 1× solutions of non-essential amino acids and sodium pyruvate (Invitrogen, Carlsbad, CA).
Splenocytes (8 × 10 5 cells) were treated with CBD (0.1-20 μM) for 30 min at 37°C, followed by cellular activation for 24 hr in complete medium containing 2% BCS in 48-well culture plates at 0.8 ml/well. Cells were activated with either 40 nM/0.5 μM PMA/Io or 100 ng immobilized anti-CD3 plus 1 μg/ml soluble anti-CD28 (BD Biosciences, San Jose, CA). Alternatively, Jurkat cells (5 × 10 4 cells) were treated with CBD (0.1-10 μM) for 30 min at 37°C, followed by cellular activation for 24 hr in complete medium containing 2% BCS in 48-well culture plates at 0.25 ml/well. Jurkat cells were activated with 40 nM/0.5 μM PMA/Io. Cells were harvested and supernatants were collected and assayed for human IL-2, or murine IL-2 or IFN-γ production by ELISA. Recombinant purified human IL-2 or mouse IL-2 or IFN-γ (BD Biosciences, San Jose, CA) served as standards from which the amount of cytokine in the samples could be determined. Capture antibodies were purified anti-human IL-2 or anti-mouse IL-2 or IFN-γ and detection antibodies were biotinylated anti-human IL-2 or anti-mouse IL-2 or IFN-γ (BD Biosciences, San Jose, CA). Color development was performed using streptavidin peroxidase followed by tetramethylbenzidine (Fluka/Sigma, St. Louis, MO). Reactions were stopped with 6N H2SO4, after which samples were read at 450 nm.
2.5 Real time polymerase chain reaction (PCR)
Splenocytes (5 × 10 6 cells) were treated with CBD (0.5-10 μM) for 30 min at 37°C, followed by cellular activation for 6 hr in complete medium containing 2% BCS in 6-well culture plates at 5 ml/well. Cells were activated with 40 nM/0.5 μM PMA/Io. Cells were harvested and placed in TRI Reagent (Sigma, St. Louis, MO). Following phase separation with bromochlorophenol, RNA was precipitated from the aqueous phase with isopropanol. The remainder of the extraction, purification and DNase treatment was done using the Promega SV Total RNA Isolation System (Promega, Madison, WI). Total RNA was reversed transcribed using random primers with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). cDNA was amplified with Taqman primers and probe sets purchased from Applied Biosystems and analyzed using a 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA).
2.6 Immunofluorescence analysis
Splenocytes (8 × 10 5 cells) were treated with CBD (0.2-20 μM) for 30 min at 37°C, followed by cellular activation with 40 nM/0.5 μM PMA/Io for 24 hr in complete medium containing 2% BCS in 48-well culture plates at 0.8 ml/well. Cells were harvested, stained with antibodies directed against CD3 or CD25 (CD3-FITC or CD25-PE; BD Biosciences, San Jose, CA), and analyzed using a FACSCalibur (BD Biosciences, San Jose, CA). Cells were gated based on forward and side scatter (FSC/SSC) and data were analyzed using CellQuest software (BD Biosciences, San Jose, CA).
2.7 Lymphoproliferation assays
Splenocytes (2 × 10 5 cells) were treated with CBD (0.2-20 μM) for 30 min at 37°C, followed by cellular activation in complete medium containing 2% (48 hr cultures) or 5% (72 hr cultures) BCS in 96-well culture plates at 0.2 ml/well. Cells were activated with either 40 nM/0.5 μM PMA/Io, 100 ng immobilized anti-CD3 plus 1 μg/ml soluble anti-CD28, or 10 μg/ml lipopolysaccharide (LPS). Splenocytes that were activated with LPS were cultured for 72 hr; splenocytes that were activated with PMA/Io or anti-CD3/CD28 were cultured for 48 hr. Cultures were pulsed with 1 μCi/well of [ 3 H]-thymidine 18 hr prior to harvest, and the cells were harvested onto glass fiber filters using a PHD cell harvester (Cambridge Technology, Inc., Watertown, MA). Tritium incorporation was measured using a Packard Tri-Carb 2100TR Liquid Scintillation Analyzer (Packard Biosciences/Perkin-Elmer, Wellesley, MA).
2.8 Mixed lymphocyte response (MLR)
Splenocytes (1 × 10 5 cells) were treated with CBD (0.2-20 μM), followed by cellular activation with mitomycin C-treated non-self (DBA/2) splenocytes in complete medium containing 5% FBS in 96-well round bottom culture plates at 0.2 ml/well. The DBA/2 splenocytes were treated with 40 μg/ml mitomycin C for 60 min at 37°C, washed 4 times with RPMI and adjusted to the appropriate cell density such that the stimulator:responder ratio was 4:1 (4 × 10 5 mitomycin C-treated DBA/2 splenocytes: 1 ×10 5 B6C3F1 splenocytes). Cells were cultured for 96 hr. 18 hr prior to harvest, cultures were pulsed with 1 μCi/well of [ 3 H]-thymidine and tritium incorporation was measured as described above.
2.9 Transient transfections
Jurkat cells (5 × 10 5 cells) were transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) in complete medium containing 2% BCS. NFAT-luc, AP-1-luc and pTA-luc plasmids were purchased from Clontech (Mountain View, CA). Briefly, for every 5 × 10 5 cells to be transfected, pooled cells were incubated with 1.5 μg plasmid DNA and 3 μl Lipofectamine 2000 reagent, each delivered in 50 μl RPMI 1640. Plasmid DNA and Lipofectamine 2000 reagent were incubated in RPMI 1640 for 5 min, combined, and allowed to complex for 20 min prior to addition to cells. Transfected, pooled cells were then distributed to 48-well plates at 1 ml/well. Three hours post-transfection and plating, cells were treated with CBD (1-20 μM) for 30 min at 37°C, followed by cellular activation with 40 nM/0.5 μM PMA/Io for 24 hr. Supernatants were harvested and assessed for IL-2, and the cells were assessed for luciferase activity.
2.10 Luciferase assays
Luciferase activity was determined using the Luciferase Assay System and Reporter Lysis Buffer (RLB) from Promega (Madison, WI). Briefly, cells were washed once in PBS, then resuspended in 50 μl RLB per 5×10 5 cells. Cells were then frozen at -80°C for 10 min, thawed and directly transferred to opaque 96-well plates for luciferase activity determination. Luciferase substrate (100 μl) was added to each well using the Bio-Tek Synergy HT instrument with KC4 version 3.4 software (Winooski, VT). After a 2 sec delay, luciferase activity was detected over a 10 sec period and data are presented as relative light units (RLU) in counts per second (CPS). Protein determinations were performed using a Bicinchoninic Acid Assay (BCA; Sigma, St. Louis, MO).
2.11 T cell purifications
T cells were purified from whole splenocyte preparations using the Pan T Cell Isolation Kit according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Briefly, spleens were collected and a single cell suspension was generated in MACS buffer (PBS, 0.5% BSA and 2 mM EDTA). Cells were then incubated with antibody cocktail and magnetic beads that allow for negative selection of T cells in the presence of a magnetic column. Purified T cells were subsequently used for ELISA analysis and proliferation. Purity of T cells was determined using immunofluorescence analysis with antibodies directed against CD3, and generally exceeded 98%.
2.12 In vitro antibody forming cell response (AFC)
Splenocytes (2.5-5 × 10 6 cells) were treated with CBD (1-20 μM), followed by cellular activation in complete medium containing 10% heat-inactivated BCS in 48-well culture plates at 0.5 ml/well. Cells were activated with either 6.5 × 10 6 sheep erythrocytes (sRBC; Colorado Serum, Denver, CO) or 100 μg/ml LPS. Splenocytes were cultured (sRBC, 5 days; LPS, 3 days) in a Bellco stainless steel tissue culture chamber pressurized to 5.5 psi with a blood-gas mixture containing 10% O2, 7% CO2, and 83% N2. The culture chamber was incubated at 37°C and rocked continuously for the duration of the culture period. Enumeration of the antibody forming cells was based on the Jerne plaque assay [16, 17]. Briefly, 50 or 100 μl aliquots of the cultured splenocytes were combined with 0.5% melted agar (Difco/BD, Franklin Lakes, NJ), guinea pig complement (Gibco/Invitrogen, Carlsbad, CA) and sheep erythrocytes, plated, covered with a 24 × 50 mm glass cover slip, and allowed to solidify. Plates were incubated for at least 3 hr at 37°C, after which AFCs were enumerated at 6.5× magnification using a Bellco plaque viewer (Bellco Glass Co., Vineland, NJ).
2.13 In vivo antibody forming cell response (AFC)
Mice (B6C3F1, 5 per treatment group) were administered CBD (25, 50 or 100 mg/kg) or THC (50 mg/kg) in corn oil by oral gavage for 3 days. On the second day, mice were sensitized with 5 × 10 8 sRBC per mouse by i.p. injection. Four days after sRBC sensitization, mice were sacrificed and total body, spleen, thymus and kidney weights were recorded. Single cell suspensions of splenocytes were then generated and used to determine the in vivo AFC response as described above.
2.14 Statistical analysis
The mean ± S.E. was determined for each treatment group. Differences between means were determined with a parametric analysis of variance. When significant differences were detected, treatment groups were compared to the appropriate control using Dunnett’s two-tailed t test. Following a two-way analysis of variance, all groups were compared using Bonferroni’s test. A two-tailed t test was used to determine statistical significance between stimulated groups in the anti-sRBC IgM AFC response between C57BL/6 and CB1 -/- /CB2 -/- mice. Statistical analyses were performed using GraphPad Prism version 4.0a for Macintosh OS X, GraphPad Software (San Diego, CA).
3.1 CBD suppressed cytokine production in PMA/Io-stimulated splenocytes in a CB1 and CB2 receptor-independent manner
Previous work from our laboratory demonstrated that CBD was one of the most potent cannabinoids for suppression of PMA/Io-induced IL-2 production in splenocytes . As seen in Figure 1B , CBD also suppressed PMA/Io-induced IFN-γ production, although the potency with which CBD suppressed IFN-γ was not as marked as for IL-2 (shown in Figure 1A as a comparative control). The CBD-induced suppression of both cytokines occurred at the level of mRNA ( Figure 1C and D ). With the demonstration that IL-2 is a sensitive target of suppression by CBD, we next determined the effect of CBD on expression of the IL-2 receptor α chain (CD25). CBD suppressed cell surface expression of CD25 in a concentration-dependent manner in PMA/Io-stimulated splenocytes ( Figure 2 ). Interestingly, there was not a large population of CD25 + cells in the absence of stimulation, suggesting the primary effect of CBD on CD25 occurs during T cell activation as opposed to an effect on the T regulatory cell population. Finally, there was no difference in the ability of CBD to suppress PMA/Io-stimulated IL-2 and IFN-γ from splenocytes derived from either C57BL/6 wild type or CB1 -/- /CB2 -/- mice ( Figure 3 ).
CBD suppressed cytokine production in PMA/Io-stimulated B6C3F1 splenocytes. A-B.) Splenocytes (8 × 10 5 cells) were treated with CBD (0.1-15 μM) for 30 min, followed by cellular activation with PMA/Io for 24 hr. Supernatants were harvested and the amount of IL-2 (A.) or IFN-γ (B.) was determined by ELISA. The data are expressed as the mean Units/ml ± SE of triplicate cultures. C-D.) Splenocytes (5 × 10 6 cells) were treated with CBD (0.5-10 μM) for 30 min, followed by cellular activation with PMA/Io for 6 hr. Real time PCR was performed for IL-2 and IFN-γ. Fold change was calculated as compared to NA. * or ** denotes values that are significantly different from the vehicle control at p < 0.05 or 0.01. Results are representative of at least two separate experiments. NA, naïve (untreated); VH, vehicle (0.1% ethanol).
CBD suppressed CD25 cell surface expression in PMA/Io-stimulated B6C3F1 splenocytes. Splenocytes (8 × 10 5 cells) were treated with CBD (0.2-20 μM) for 30 min, followed by cellular activation with PMA/Io for 24 hr. Cells were harvested and stained with fluorescent antibodies directed against CD3 (FITC) or CD25 (PE). Cells were gated on FSC/SSC. Numbers denote percent gated events. Results are representative of three separate experiments. NA, naïve (untreated); VH, vehicle (0.1% ethanol).
CBD suppressed cytokine production in wild type C57BL/6 and CB1 -/- /CB2 -/- splenocytes. Splenocytes (8 × 10 5 cells) were treated with CBD (0.2-10 μM) for 30 min, followed by cellular activation with PMA/Io for 24 hr. Supernatants were harvested and the amount of IL-2 (A-B.) or IFN-γ (C-D.) in wild type C57BL/6 and CB1 -/- CB2 -/- was determined by ELISA. The data are expressed as the mean Units/ml ± SE of triplicate cultures. * or ** denotes values that are significantly different from the respective vehicle control at p < 0.05 or 0.01. Data are presented as % VH control in B and D. Results are representative of two separate experiments. NA, naïve (untreated); VH, vehicle (0.1% ethanol).
3.2 CBD suppressed cytokine production in T cells
In order to determine whether splenic T lymphocytes are direct targets of inhibition by CBD, splenocytes were activated with anti-CD3/anti-CD28, which exclusively stimulates T lymphocytes via the T cell receptor. Although not as marked as the inhibition of cytokines produced in response to PMA/Io, CBD also suppressed IL-2 and IFN-γ produced in response to anti-CD3/anti-CD28 from splenic T lymphocytes ( Figures 4A and B ). Furthermore, PMA/Io-induced IL-2 and IFN-γ production from purified splenic T cells (i.e., >95% purity) was also suppressed by CBD ( Figures 4C and D ). It is noteworthy that purified T cells were particularly sensitive to CBD in the presence of PMA/Io and therefore, lower concentrations of CBD were used for these studies.
CBD suppressed cytokine production in B6C3F1 splenic T cells. A-B.) Splenocytes (8 × 10 5 cells) were treated with CBD (0.1-15 μM) for 30 min, followed by cellular activation with immobilized anti-CD3 plus soluble anti-CD28 for 24 hr. Supernatants were harvested and the amount of IL-2 (A.) or IFN-γ (B.) was determined by ELISA. C-D.) T cells purified from splenocytes (8 ×10 5 cells) were treated with CBD (0.1-2 μM) for 30 min, followed by cellular activation with PMA/Io for 24 hr. Supernatants were harvested and the amount of IL-2 (C.) or IFN-γ (D.) was determined by ELISA. The data are expressed as the mean Units/ml ± SE of triplicate cultures. * or ** denotes values that are significantly different from the vehicle control at p < 0.05 or 0.01. Results are representative of at least two separate experiments. NA, naïve (untreated); VH, vehicle (0.1% ethanol).
3.3. CBD suppressed cellular proliferation in several cell types
IL-2, which acts via the IL-2 receptor, is a critical cytokine for T cell proliferation; therefore we determined the effect of CBD on cellular proliferation. CBD suppressed PMA/Io-stimulated proliferation in a concentration-dependent manner ( Figure 5A ). However, as PMA/Io likely induces proliferation in most splenic cell types, various stimuli were utilized to target specific cell populations. Cellular proliferation in response to LPS, which predominantly activates B cells, was also suppressed in a concentration-dependent manner by CBD ( Figure 5B ). In order to address the effect of CBD on T cell proliferation, two different stimuli were used: anti-CD3/anti-CD28, or mitomycin C-treated allogeneic lymphocytes (MLR). In response to either anti-CD3/anti-CD28 or mitomycin C-treated allogeneic lymphocytes, both of which activate T cells via the T cell receptor, CBD suppressed T cell proliferation ( Figures 5C and D ).
CBD suppressed cellular proliferation in B6C3F1 splenocytes in response to various stimuli. A-D.) Splenocytes (2 × 10 5 cells) were treated with CBD (0.2-10 μM) for 30 min, followed by cellular activation. 18 hours prior to harvest, cells were pulsed with 1 μCi 3 H-thymidine. Cells were harvested onto glass fiber filters and tritium incorporation was measured with a liquid scintillation counter. Splenocytes were activated with A.) PMA/Io for 48 hr; B.) LPS for 72 hr; C.) immobilized anti-CD3 plus soluble anti-CD28 for 48 hr; D.) mitomycin C-treated allogeneic lymphocytes for 96 hr. The data are expressed as the mean CPM ± SE of quadruplicate cultures. * or ** denotes values that are significantly different from the vehicle control at p < 0.05 or 0.01. Results are representative of at least three separate experiments. R, responders; S, stimulators; NA, naïve (untreated); VH, vehicle (0.1% ethanol).
3.4 CBD suppressed the T cell-dependent AFC response
One functional immune endpoint that is sensitive to suppression by other plant-derived and synthetic cannabinoids is the T cell-dependent AFC response [18, 19]. As seen in Figure 6A , and consistent with THC, CBD suppressed the in vitro T cell-dependent anti-sRBC IgM AFC response, but did not affect the in vitro IgM AFC response to the polyclonal B cell activator, lipopolysaccharide (LPS; Figure 6B ).
Effect of CBD on the IgM AFC response in B6C3F1 splenocytes. A-B.) Splenocytes (5 × 10 6 cells for sRBC; 2.5 × 10 6 cells for LPS) were treated with CBD (1-20 μM) or THC (20 μM) for 30 min, followed by stimulation with A.) sRBC for 5 days or B.) LPS for 72 hr. Cells were then incubated in a Bellco stainless steel tissue culture chamber pressurized to 5.5 psi with a gas mixture consisting of 10% O2, 7% CO2 and 83% N2. The culture chamber was placed at 37°C with constant rocking for the duration of the culture period. Enumeration of the AFC response was performed as described in Materials and Methods. The data are expressed as the mean AFC/10 6 viable cells ± SE of quadruplicate cultures. C.) B6C3F1 mice received CBD (25-100 mg/kg) or THC (50 mg/kg) by oral gavage for 3 days. On day 2, in addition to drug, mice received a single i.p. injection of sRBC (5 × 10 8 cells/mouse). Mice were sacrificed on day 6, after which the AFC response was enumerated as described in Materials and Methods. The data are expressed as the mean AFC/10 6 viable or recovered cells ± SE. Results are pooled from two separate experiments. * or ** denotes values that are significantly different from the vehicle control at p < 0.05 or 0.01.
As CBD suppressed the in vitro anti-sRBC IgM AFC response, the effect of CBD in vivo was determined. Oral administration of CBD produced a modest modulation of the IgM AFC response to sRBC ( Figure 6C ). Although none of the CBD treatments produced statistically significant modulation (at p < 0.05), the trend for CBD-induced suppression of the in vivo AFC response to sRBC at 100 mg/kg was consistent in two separate replicates of the experiment. The magnitude of suppression by 100 mg/kg CBD was similar to that produced by 50 mg/kg THC. There was no significant change in total body weight, spleen or thymus weights, or in the weight of the kidneys, presumably non-targets of immunosuppression by cannabinoids (data not shown).
Although CBD has been reported to possess low affinity for both CB1 and CB2 cannabinoid receptors [2, 3], we next attempted to discern and/or confirm the lack of a role for CB1 and CB2 in CBD-induced suppression of the anti-sRBC IgM AFC response by using splenocytes derived from CB1 -/- /CB2 -/- mice. As seen in Figure 7 , CBD robustly suppressed the anti-sRBC IgM AFC response in C57BL/6 mice. There was a significant decrease (p < 0.001) in the overall magnitude of AFC induced by sRBC in the CB1 -/- /CB2 -/- mice versus C57BL/6 mice (379.7 ± 65.7 versus 3829 ± 443, respectively, over 4 separate experiments; one representative graph depicted in Figure 7A ). Since there was such a large discrepancy in the magnitude of the immune response to sRBC in the CB1 -/- /CB2 -/- mice, the data are also expressed as percent of vehicle control for four separate experiments ( Figure 7B ). There was no difference in CBD-induced suppression of the anti-sRBC IgM response in either genotype with the exception of the 5 μM concentration, which was highly variable in the CB1 -/- CB2 -/- mice. It is likely that CB1 and/or CB2 are not involved in CBD-induced suppression of the in vitro anti-sRBC IgM AFC response. On the other hand, it is evident that either CB1 or CB2, or both, contribute to the overall magnitude of the in vitro anti-sRBC IgM AFC response.
Effect of CBD on the in vitro AFC response in wild type C57BL/6 and CB1 -/- /CB2 -/- splenocytes. Splenocytes (5 × 10 6 cells) from C57BL/6 or CB1 -/- /CB2 -/- were treated with CBD (1-20 μM) or THC (20 μM) for 30 min, followed by stimulation with sRBC for 5 days. Cells were then cultured and the AFC were enumerated as stated in Figure 1 . The data are expressed as the mean AFC/10 6 viable cells ± SE of quadruplicate cultures for one representative experiment (A.) or % VH results for CBD from four experiments are pooled, with the exception of the 15 μM group, which represents a single experiment (B.). * or ** denotes values that are significantly different from the respective vehicle control at p < 0.05 or 0.01.
3.5 CBD suppressed NFAT and AP-1 reporter gene activity in PMA/Io-stimulated Jurkat cells
CBD suppressed IL-2 and IFN-γ cytokine production in various T cell preparations, both of which are regulated by several transcription factors, including AP-1 and NFAT [20-23]. Interestingly, both AP-1 and NFAT have been shown to be sensitive targets of inhibition by many cannabinoids [24-26]. Thus, we determined whether NFAT and AP-1 were also targeted by CBD using AP-1- and NFAT-driven luciferase reporter genes in human Jurkat T cells. As shown in Figure 8A , CBD did suppress human IL-2 production from PMA/Io-stimulated Jurkat T cells. Furthermore, the mechanism involves suppression of transcription as CBD suppressed PMA/Io-induced AP-1- and NFAT-luciferase expression in a concentration dependent manner ( Figure 8B and 8C , respectively). There was no effect on the overall protein content (data not shown) in the various treatment groups, indicating, in fact, that the suppression of luciferase activity was due to CBD. Consistent with other cannabinoids , suppression of NFAT-luciferase was more robust than AP-1-luciferase.
CBD suppressed IL-2 production and AP-1 and NFAT activity in PMA/Io-stimulated human Jurkat T cells. A.) Jurkat cells (5 × 10 4 cells) were treated with CBD (0.1-10 μM) for 30 min, followed by cellular activation with PMA/Io for 24 hr. Supernatants were harvested and the amount of IL-2 was determined by ELISA. The data are expressed as the mean Units/ml ± SE of triplicate cultures. B-C.) Jurkat cells (5 × 10 5 cells) were transiently transfected with either AP-1-luciferase (B.) or NFAT-luciferase (C.). Three hours later, cells were treated with CBD (1-10 μM) for 30 min, followed by cellular activation with PMA/Io for 21 hr. Luciferase activity was determined as described in Materials and Methods. The data are expressed as the mean CPS of triplicate cultures. * or ** denotes values that are significantly different from the vehicle control at p < 0.05 or 0.01. Results are representative of at least two separate experiments. NA, naïve (untreated); VH, vehicle (0.1% ethanol).
CBD suppressed several immunological endpoints, with a profile of activity similar to other plant-derived, synthetic and endogenous cannabinoids [9, 18, 27-29]. Specifically, CBD suppressed cytokine production from activated primary mouse splenocytes in a concentration-dependent manner. Of note was the observation that this cytokine suppression occurred independently of either CB1 or CB2 as demonstrated in splenocytes derived from CB1 -/- /CB2 -/- mice. The major advantages of evaluating immunological endpoints using CB1 -/- /CB2 -/- mice, rather than the currently available CB1 and CB2 antagonists, are the absence of potential inverse agonism and/or direct effects of the antagonists, as has been reported [30, 31]. Despite potential activity with antagonists under certain conditions, the antagonists have been used to suggest that CBD might exert some of its effects via CB1 and/or CB2 . There is also recent evidence that CBD might exert its effects via a yet unidentified cannabinoid receptor or the newly identified putative cannabinoid receptor, GPR55 . Furthermore, there are reports that CBD binds the vanilloid receptor, VR1 , is an agonist at the serotonergic 5HT-1a receptor [34, 35], and is an agonist at A2a receptors in microglial cells . In addition, Drysdale, et. al. demonstrated that the CBD-induced elevation in intracellular calcium in hippocampal cells was exacerbated in the presence of either a CB1 receptor antagonist or a VR1 receptor antagonist, suggesting signaling interactions between CBD and these receptors through an unknown mechanism . Although we demonstrate that CBD-induced suppression of cytokine production from PMA/Io-stimulated splenocytes was independent of CB1 and CB2, we cannot exclude the possibility that CBD exerts its effects through one or more of the other aforementioned receptors at this time.
In addition to suppression of PMA/Io-stimulated IL-2 production, CBD suppressed IFN-γ production. The effect of CBD was more robust on IL-2 than IFN-γ from PMA/Io-stimulated splenocytes. However, in splenocytes that were stimulated through the T cell receptor using anti-CD3/anti-CD28, CBD exhibited similar potency on suppression of IL-2 and IFN-γ. The difference in sensitivity of CBD-induced suppression of IL-2 might be associated with the rapid elevation of intracellular calcium seen with PMA/Io, with PMA/Io-stimulated IL-2 being more sensitive to a rapid calcium rise than anti-CD3/anti-CD28-stimulated IL-2. This is supported by our previous observation that pretreatment of splenocytes with agents that elevate intracellular calcium resulted in suppression of PMA/Io-stimulated IL-2 , whereas the endogenous cannabinoid 2-arachidonoyl-glycerol (2-AG)-induced suppression of PMA/Io-stimulated IFN-γ could be partially reversed by increasing intracellular calcium . The sensitivity of both IL-2 and IFN-γ, however, does suggest that there exists a common target of inhibition by CBD. One likely common target might be NFAT since NFAT is critical for both IL-2 and IFN-γ , and because NFAT DNA binding activity is markedly decreased in activated T cells when cultured in the presence of two other cannabinoids, CBN or 2-AG [24-26]. Indeed, CBD suppressed AP-1- and NFAT-luciferase gene expression. Although the CBD-induced suppression of NFAT-luciferase gene expression was more robust, it is notable that AP-1 proteins bind cooperatively with NFAT at many NFAT responsive elements in both IL-2 and IFN-γ [21, 23].
The demonstration that CBD suppresses NFAT activity is further supported by our observation that CBD suppressed cell surface expression of CD25, which is also regulated by NFAT . Interestingly, this effect appeared to be T cell-specific. Although CBD suppressed cell surface expression of CD25 on the CD3 + population, there was no effect on the CD3 – (i.e., non-T cell, and presumably, mainly B cell) population. These results were also consistent with the observation that CBD suppressed the IgM AFC response to the T cell-dependent antigen, sRBC, but had no effect on the IgM AFC response to the polyclonal B cell activator, LPS. This is in contrast to our observation that CBD also suppressed LPS-induced proliferation, which has been classically identified as a B cell response. However, there is much evidence to suggest that T cells will proliferate in response to LPS and that T cells express the appropriate toll-like receptors important for this response [39, 40]. Alternatively, these results suggest that the mechanism of immunosuppression by CBD, and likely other cannabinoids for which this dichotomy has been observed , might involve a generalized suppression of cellular proliferation to certain stimuli. Overall, the ability of CBD to suppress PMA/Io-, LPS-, anti-CD3/anti-CD28-induced proliferation, in combination with suppression of the MLR, provides more evidence that CBD targets T cells.
The profile with which CBD targeted T cell cytokine production and proliferation was very similar to that previously reported for two other plant-derived cannabinoid compounds, THC and CBN [18, 19]. These results suggest that the mechanisms by which these three plant-derived cannabinoid compounds suppress cytokine production, at least in vitro, is similar and does not require CB1 or CB2. However, CBD only modestly suppressed the in vivo anti-sRBC IgM AFC response. These data support previous studies conducted in male CD-1 mice in which CBD (≤ 25 mg/kg) did not suppress the in vivo anti-sRBC IgM AFC response . With the demonstration that CBD is efficacious in vivo in a variety of model systems [6, 7, 15, 42], these results suggest that the AFC response in vivo is rather refractory to inhibition by CBD. One explanation for the absence of suppression of the in vivo AFC response is that CBD might be rapidly metabolized in vivo to a non-functional metabolite, particularly because CBD was administered orally. The discrepancy between CBD and THC in vivo versus in vitro does implicate the involvement of one or both cannabinoid receptors in the anti-sRBC IgM AFC response. Further evidence of the requirement for CB1 and/or CB2 in the in vivo anti-sRBC IgM AFC is provided by our observations that the THC-induced suppression of this response was abolished in CB1 -/- /CB2 -/- mice (Springs, et. al., submitted for publication), suggesting that cannabinoids must possess affinity for either CB1 or CB2 to suppress the AFC response in vivo.
We also attempted to discern the role of CB1 and CB2 in CBD-induced suppression of the in vitro anti-sRBC IgM AFC response using splenocytes derived from CB1 -/- /CB2 -/- mice. Interestingly, the control anti-sRBC IgM AFC response by splenocytes isolated from CB1 -/- /CB2 -/- mice was remarkably low as compared to that observed with splenocytes isolated from wild type C57BL/6 mice, which limited our ability to absolutely conclude whether CB1 and/or CB2 played a critical role in CBD-induced suppression of the in vitro anti-sRBC IgM AFC response. However, upon examination of the data as percent of vehicle control, it appears unlikely that CB1 and/or CB2 are involved. Although there was a difference between genotypes at lower concentrations of CBD, the response was highly variable, which is likely related to the degree to which the CB1 -/- /CB2 -/- cells were stimulated in vitro with sRBC.
The above results also strongly suggest that CB1 and/or CB2 play a critical role in the in vitro anti-sRBC IgM AFC response. Currently, the reasons underlying the marked difference in the magnitude of the immune response to sRBC between splenocytes derived from CB1 -/- /CB2 -/- and wild type C57BL/6 mice remain to be fully elucidated. Part of the mechanism might involve compromised accessory cell function (eg. macrophages) in CB1 -/- /CB2 -/- mice since there was no difference in the magnitude of the in vitro IgM AFC response by purified B cells activated with CD40 ligand-expressing L cells, a stimulation which does not require T cells or macrophages (Springs, et. al., submitted for publication). Notably, no difference in the magnitude of the in vivo anti-sRBC IgM AFC response between CB1 -/- /CB2 -/- and wild type C57BL/6 mice was observed (Springs, et. al., submitted for publication), suggesting the existence of additional compensatory mechanisms in vivo.
Overall, CBD did not significantly alter the anti-sRBC IgM AFC response in vivo, but CBD did suppress a number of immune responses in vitro, specifically involving T cells as primary effectors. Using splenocytes derived from CB1 -/- /CB2 -/- mice, it was determined that CBD-induced suppression of cytokine production and suppression of the in vitro anti-sRBC IgM AFC response was CB1 and CB2 receptor-independent. More importantly, the observation of the significant difference in the control anti-sRBC IgM AFC response in CB1 -/- /CB2 -/- mice as compared to wild type C57BL/6 mice suggests that CB1 and/or CB2 play a critical role in the magnitude of the response to sRBC in vitro. These data provide a detailed characterization of CBD’s effects on various immunological endpoints and provide further evidence that the mechanisms by which cannabinoids modulate immune function is both cannabinoid receptor-dependent and – independent. Finally, these data demonstrate that transcription factors in the IL-2 promoter are sensitive targets of inhibition by CBD.
We would like to thank Dr. Andreas Zimmer (University of Bonn) for kindly providing the CB1 -/- /CB2 -/- knockout mice. We would also like to thank Mr. Robert Crawford and Ms. Amber Crawford for excellent technical assistance and Mrs. Kimberly Hambleton for assistance in the preparation of the manuscript.
Immune Responses Regulated by Cannabidiol
Department of Basic Sciences, Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi.
Barbara L.F. Kaplan
Department of Basic Sciences, Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi.
Department of Basic Sciences, Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, Mississippi.
* Address correspondence to: Barbara L.F. Kaplan, Department of Basic Sciences, Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, 240 Wise Center Drive, Mississippi State, MS 39762 [email protected]
This Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are cited.
Introduction: Cannabidiol (CBD) as Epidiolex ® (GW Pharmaceuticals) was recently approved by the U.S. Food and Drug Administration (FDA) to treat rare forms of epilepsy in patients 2 years of age and older. Together with the increased societal acceptance of recreational cannabis and CBD oil for putative medical use in many states, the exposure to CBD is increasing, even though all of its biological effects are not understood. Once such example is the ability of CBD to be anti-inflammatory and immune suppressive, so the purpose of this review is to summarize effects and mechanisms of CBD in the immune system. It includes a consideration of reports identifying receptors through which CBD acts, since the “CBD receptor,” if a single one exists, has not been definitively identified for the myriad immune system effects. The review then provides a summary of in vivo and in vitro effects in the immune system, in autoimmune models, with a focus on experimental autoimmune encephalomyelitis, and ends with identification of knowledge gaps.
Conclusion: Overall, the data overwhelmingly support the notion that CBD is immune suppressive and that the mechanisms involve direct suppression of activation of various immune cell types, induction of apoptosis, and promotion of regulatory cells, which, in turn, control other immune cell targets.
Cannabidiol History and Therapeutic Uses
Cannabidiol (CBD) is a plant-derived cannabinoid that has structural similarity to the primary psychotropic congener in cannabis, Δ 9 -tetrahydrocannabinol (THC). While CBD was initially isolated in the 1940s, its structure was not elucidated until the 1960s. 1,2 Unlike THC, CBD is bicyclic, comprised a terpene and an aromatic ring, and is a pentyl side chain. 1 It exists as two enantiomers, and it is (−)CBD 3 that is one of the major constituents found in Cannabis sp., and will be the focus of this review. For many years, THC and CBD were designated as psychoactive and nonpsychoactive, respectively, owing to the fact that THC produces the euphoric high associated with cannabis use, while CBD does not. However, since we know that CBD produces biological effects in the central nervous system (CNS), perhaps it is better defined as psychoactive, but not psychotropic, since it is active in the CNS without producing the euphoric high.
Perhaps it was the association of the euphoric high with THC that provided the initial focus on THC as opposed to CBD for potential medical use, since THC was originally identified as the active component of the plant. 4 However, in recent years, researchers have begun to explore CBD more as a therapeutic addition or alternative to THC. In the United States, oral THC (dronabinol, Marinol ® ) was first approved in 1985 by the Food and Drug Administration (FDA) to treat nausea and vomiting associated with chemotherapy. In 1992, dronabinol was also approved to treat cachexia in AIDS patients. 5 The next major advancement in cannabinoid pharmaceuticals was not until the mid-2000s when Sativex ® (nabiximols), a combination of THC and CBD as an oromucosal spray, was approved in Canada and the EU for neuropathic pain in multiple sclerosis (MS) and intractable cancer pain. 6 There are several reasons why combining THC and CBD in a single therapeutic could have value. 6 First, additional therapeutic benefit might be gained from hitting multiple targets; for example, if THC alleviates pain and CBD alleviates anxiety, 7–16 the combination therapy could be quite effective for chronic pain sufferers. Second, for disease states in which both THC and CBD are efficacious, a combination might allow for lower doses of THC, thereby potentially decreasing the psychotropic effects of THC. Third, there are some studies suggesting pharmacokinetic interactions between CBD and THC in which CBD treatment increases THC levels, 17–20 thereby allowing longer duration of effects of THC. Sativex ® has been evaluated in several clinical trials for spasticity associated with MS, neuropathic pain, and other conditions. 21–37
The latest approved cannabinoid pharmaceutical in the United States is CBD as Epidiolex ® . It was approved by the U.S. FDA in 2018 for epilepsy in children, in particular, for Dravet Syndrome and Lennox-Gastaut Syndrome. 38–42 CBD is also being investigated for its effectiveness in other diseases, including Tuberous Sclerosis, a genetic condition that causes growth of benign tumors all over the body, 43,44 schizophrenia, 45 and refractory epileptic encephalopathy. 46
In addition to the federally approved uses of CBD as Epidolex ® , CBD, usually as CBD oil, is widely used for putative medical benefit in several states, and is certainly used in states in which cannabis has been decriminalized, or legalized, for recreational use. 47 There are reports that CBD and other cannabinoids are beneficial for sleep, anxiety, pain, post-traumatic stress disorder, schizophrenia, neurodegenerative disorders, and immune-mediated diseases. 48 Often these conditions are self-diagnosed and self-treated, so there can be issues with dosing, other drug interactions, and characterization of CBD safety and efficacy.
Overall, it is clear that exposures to CBD are increasing. 47,49–51 It is also clear that CBD possesses therapeutic benefit, and in some cases, the beneficial effects of CBD are for diseases for which other available treatments have not been efficacious. 52 Together, these observations demonstrate the critical need to continue research on CBD, and therefore the goal of this review is to provide a summary of the effects and mechanisms by which CBD alters immune function. The review will include an evaluation of the role for various receptors through which CBD acts in the immune system. There will also be a description of CBD effects in animal and human immune responses, a characterization of mechanisms by which CBD mediates immune effects, and identification of knowledge gaps regarding CBD’s actions in the immune system.
Identification of CBD Receptors and Other Targets
Upon identification of the cannabinoid receptors, CBD was determined to exhibit low affinity for CB1 53 and CB2 receptors. 54 Consistent with this, we showed CBD-induced suppression of cytokine production in mouse splenocytes in both wild-type and double cannabinoid receptor knockout mice (Cnr1 −/− /Cnr2 −/− mice). 55 Another study demonstrated that ophthalmic administration of CBD following corneal inflammation reduced neutrophils in both wild-type and CB2 receptor knockout mice. 56 CBD-mediated suppression of anti-CD3-mediated proliferation of T cells also occurred in both wild-type and CB2 receptor knockout splenocytes. 57 However, there are a few reports using inflammatory stimuli in which CBD’s actions have been attributed to either CB1 or CB2 receptors ( Table 1 ). In a sepsis model induced with bacterial lipopolysaccharide (LPS), CBD-mediated inhibition of gastric emptying was reversed with the CB1 receptor antagonist, AM251. 58 Similarly, CBD inhibited interleukin (IL)-1 in a hypoxia-ischemia brain insult model and this effect was reversed with the CB2 receptor antagonist, AM630. 59 Use of ovalbumin to induce an asthma-like disease in mice demonstrated that some cytokines and chemokines induced in the lungs of mice that were suppressed by CBD (IL-4, IL-5, IL-13, and eotaxin) were differentially regulated by CB receptors. 60 Specifically, CBD-induced suppression of IL-5 was reversed in the presence of the CB2 receptor antagonist in bronchoalveolar lavage fluid and lung tissue, but there was no clear receptor dependence identified for CBD’s suppression of IL-4, IL-13, or eotaxin. 60 Thus, several studies do suggest a possible role for cannabinoid receptors in CBD-mediated suppression of inflammatory effects. It should also be noted that there are several reports suggesting that CBD acts as an allosteric modulator of CB1 or CB2 receptors, 61–64 although the role for CB1 or CB2 receptor allosteric modulation by CBD in immune function has not yet been determined.
Receptors Identified in Mediating Cannabidiol Immune Effects
FAAH, fatty acid amide hydrolase; PPAR-γ, peroxisome proliferator-activated receptor gamma; TRPV1, transient receptor potential vanilloid 1.
Another mechanism by which CBD acts is through inhibition of fatty acid amide hydrolase (FAAH), 65–67 suggesting that some of CBD’s effects are mediated by anandamide elevation since FAAH is responsible for the breakdown of anandamide. 65,66 Anandamide is an endogenous cannabinoid that exhibits affinity for CB1 and CB2 receptors. 68,69 A recent study suggested that the mechanism by which CBD elevates anandamide involves CBD interaction with fatty acid binding proteins, which prevents anandamide binding to these proteins to block anandamide transport to FAAH. 67 Since anandamide exhibits affinity for CB1 and CB2 receptors, and oxidation products of anandamide through cyclooxygenase or cytochrome P450 enzymes produce metabolites that also exhibit affinity for CB1 and CB2 receptors, 70,71 anandamide or its metabolites could account for some of the reports that CBD acts through CB1 and/or CB2 receptors. 58,61–64,72–84
Actions of CBD in immune function might also be mediated by the transient receptor potential V1, known as the vanilloid receptor (TRPV1), which was found to be activated by CBD. 65 Specifically, CBD was found to increase intracellular calcium in HEK cells transfected with TRPV1, and the CBD-induced increase in calcium was blocked by the TRPV1 antagonist, capsazepine. 65,66 Follow-up studies demonstrated that CBD desensitizes TRPV1 following activation. 85 Other studies have suggested that CBD acts through TRPV1 in the immune system ( Table 1 ). CBD can induce myeloid-derived suppressor cells (MDSCs), a type of regulatory cell, in the liver, and this effect is lost in TRPV1 knockout mice. 86 Specifically, regarding inflammation, CBD attenuated thermal hyperalgesia in response to carrageenan injections or in a neuropathic pain model in a capsazepine-dependent manner. 87,88 CBD suppression of cytokines in inflamed primary human colonic tissue was attenuated by the TRPV1 antagonist, SB366791. 82 SB366791 was also effective in reversing CBD’s suppression of rolling and adherent leukocytes in the sodium monoiodoacetate model of osteoarthritis in rats. 83 Together, these data suggest that TRPV1 is a critical receptor through which CBD acts in the immune system.
There have been several critical articles in which adenosine A2A receptors have been shown to mediate CBD’s effects in the immune system. 89–91 CBD was shown to inhibit microglial cell proliferation, which was associated with inhibition of adenosine uptake into cells. 89 The studies also demonstrated that CBD suppression of tumor necrosis factor-alpha (TNF-α) could be reversed using an adenosine A2A receptor antagonist, and CBD-induced suppression of LPS-stimulated TNF-α was not observed in adenosine A2A receptor knockout mice. 89 The role for adenosine A2A receptor in CBD-mediated neuroprotection or suppression of neuroinflammation was demonstrated in a model of hypoxia-ischemia in newborn mouse brains. 90 CBD inhibited adenosine uptake into rat microglial cells and CBD enhanced adenosine’s ability to inhibit TNF-α, which was prevented by the adenosine A2A receptor antagonist, ZM241385. 91 These studies show that CBD acts through the adenosine A2A receptor, especially in microglial cells.
CBD’s effects have also been shown to be mediated by peroxisome proliferator-activated receptor gamma (PPAR-γ) using PPAR-γ antagonists in models of β amyloid neuroinflammation, 92 apoptosis, 93,94 dinitrobenzene sulfonic acid (DNBS)-induced colitis, 95 human ulcerative colitis, 96 LPS activation of microglial cells, 97 and hypoxia-ischemia model of neuroinflammation. 98
There are several reports that CBD acts through the serotonin 5-HT1a receptor ( Table 1 ). Although most of the evidence for the involvement of this receptor comes from the attenuation of CBD’s effects using the 5-HT1a antagonist, WAY100635, early studies demonstrated that CBD displaced binding of the 5-HT1a agonist, 8-OH-DPAT, in membranes from CHO cells expressing the human 5-HT1a receptor. 99 Few of the CBD-mediated effects acting through the serotonin 5-HT1a receptor have been reported in immune cells, but immune cells do express 5-HT1a. 100–103 One study showed that IL-1 produced in the brain in response to hypoxia-ischemia insult was inhibited by CBD, and reversed with the 5-HT1a receptor antagonist, WAY100635. 59
Studies have suggested that CBD might act through other receptors, including other TRP receptors, 66,85,104–107 or opioid receptors. 108 There is also evidence that CBD acts through blockade of GPR55, 109 and specifically that CBD modestly antagonized proinflammatory effects in human innate cells following GPR55 activation. 110 Thus, together, the current data support that immune effects of CBD are mediated through activation of CB1, CB2, TRPV1, adenosine A2A, and PPAR-γ receptors, blockade of GPR55 receptors, and FAAH inhibition.
CBD Immune System Effects and Mechanisms
Immunity is maintained through various cell types acting together to provide protection against foreign invaders, and simultaneously avoid reactions against self-proteins. Thus, an appropriate immune response requires a regulated balance between robust reactions against non-self, but limited or no reactions against self. Cell types include neutrophils, macrophages, and other myeloid cells comprising the innate immune system, which reacts quickly to destroy pathogens. In the event that an innate response is insufficient, certain innate cells can activate the adaptive immune response, comprised predominantly of T and B cells. T cells can then provide signals that recruit and activate other immune cells, or directly lyse or induce apoptosis of infected cells. T cells can also help stimulate B cells, which produce antibodies to neutralize pathogens and/or enhance destruction of the pathogens. Communication between the various cell types, and therefore the innate and adaptive immune responses, is mediated by expressed or secreted proteins called cytokines or chemokines. Inflammation is the process commonly associated with the innate immune response since pathogen destruction can also cause tissue damage, although T cells certainly are proinflammatory as well. In fact, many cell types, regardless of whether they are immune cells, produce proinflammatory cytokines in response to inflammation.
The effects of CBD on immune responses can involve innate or adaptive responses. In assessing these responses, various cell types and their functions have been examined. For instance, a common end-point to examine regardless of cell type is cytokine or chemokine production. Typical proinflammatory cytokines include IL-1α, IL-1β, IL-6, TNF-α and IL-17A, while IL-10 is considered anti-inflammatory. Some cytokines are produced by specific T cell subsets; for instance, the Th1 subset produces interferon-gamma (IFN-γ) and promotes cell-mediated cytotoxicity, while the Th2 subset produces IL-4 and promotes B cell responses. Other end-points that might provide clues of disruption of immune competence are nitric oxide or myeloperoxidase (MPO) production from innate cells, as these are often released during pathogen destruction. Thus, the effects of CBD on immune function are presented by cell type, outlining known mechanisms by which CBD alters various end-points. Tables 2–4 include the studies described in the text (and others) and are organized by experimental approach. As indicated above, inflammation can induce proinflammatory cytokine production in nonimmune cells, so there are also a few of those examples included in the tables.
Cannabidiol-Induced Immune Suppression by Cell Type in Human Cells In Vitro
|Human cell lines b||↓cytokines||186|
|Jurkat and MOLT-4 T cells b||↑apoptosis||80a|
|Human coronary artery endothelial cells||↓adhesion molecules, migration, transcription factors, nitrative stress||119a|
|Jurkat T cells b||↓cytokines, transcription factors||55a|
|THP-1 cells b||↓IDO||142|
|Human intestine||↓proteins and nitric oxide||96|
|Human liver sinusoidal endothelial cells||↓adhesion molecules||118|
|Human gingival mesenchymal stem cells||↓inflammatory genes||79|
|Caco-2 cells b||↓phosphoproteins||82a|
|Primary colonic explants||↓cytokines||82a|
|Human PBMCs||↓proliferation and cytokines||146a|
|HaCaT human keratinocytes b||↓cytokines||84|
|Human plasmacytoid dendritic cells||↓CD83 expression in HIV + dendritic cells||134a|
ROS, reactive oxygen species.
Cannabidiol-Induced Immune Suppression by Animal Cell Type In Vitro
|B6C3F1 female splenocytes||↓IL-2||196|
|EL-4 T cells a||↑apoptosis||80b|
|Mouse EOC-20 microglial cells a||↓proliferation||89b|
|BALB/c male splenocytes||↓IL-4 and IFN-γ||140b|
|B6C3F1 female splenocytes||↓IL-2 and IFN-γ||55b|
|BALB/c male thymocytes and EL-4 T cells a||↑apoptosis||150b|
|BALB/c male splenocytes||↑apoptosis||151b|
|Sprague-Dawley rat microglial cells c||↓adenosine uptake,||91b|
|BV-2 cells a||↓cytokines, ↓NF-κB activation||147b|
|Mouse brain slices c||↓cytokines||90b|
|Rat male astroglial cells||↓gliosis||92b|
|C57BL/6 male Kupffer cells||↓TNF-α||118|
|BALB/c microglial cells c||↑apoptosis||156b|
|BV-2 cells a||↓oxidative stress, ↓Ccl2||159|
|MOG-specific female T cells||↓IL-17A and IL-6||144b|
|Mouse brain endothelial cells a||↓VCAM-1 and leukocyte adhesion||164b|
|Rat astrocytes c||↓Ccl2||164b|
|RAW cells a||↓TNF-α||148|
|MOG-specific female T cells||↓cytokines||143b|
|Rat male splenocytes and mesenteric lymph nodes||↓proliferation and cytokines||146b|
|Primary mouse male and female microglial cells||↓activation||97b|
|BV-2 cells a||Alteration of circadian rhythm-associated genes||197|
|BV-2 cells a||alteration of miRNAs||161b|
|C57BL/6 or BALB/c female splenocytes||↓proliferation and cytokines||57|
c Sex not stated for cells derived from animals (or in the case of primary microglial cell isolates, not determined in newborn animals).
IFN-γ, interferon-gamma; IL, interleukin; miRNA, microRNA; MOG, myelin oligodendrocyte glycoprotein; NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-alpha; VCAM-1, vascular cell adhesion molecule-1.
Cannabidiol-Induced Immune Suppression in Animals In Vivo
|Model||Disease model||Route, dose range, and duration/frequency a||Major effects||Reference|
|Male CD-1 mice||sRBC||i.p.||Modest ↓antibody production||155|
|Male DBA/2 mice||Collagen-induced arthritis||i.p. or oral||↓disease, ↓TNF-α and IFN-γ||139b|
|2.5–20 mg/kg for i.p.|
|5–50 mg/kg for oral|
|Male ICR mice||Carrageenan-induced inflammation||ethosome (CBD in ethosomal gel)||↓inflammation||198|
|100 mg of ethosomal CBD (3%)|
|Male Wistar rats||Carrageenan-induced inflammation||Oral||↓disease, ↓prostaglandin (PGE2)||199|
|Female NOD mice||Diabetes||i.p.||↓disease incidence, ↓IL-12, TNF-α and IFN-γ, ↑IL-4||123|
|Female C57BL/6 mice||EL-4 leukemia growth||i.p.||↑apoptosis of tumor cells||80b|
|12.5 or 25 mg/kg once|
|Male Wistar rats||Sciatic nerve pain or CFA-induced inflammation||Oral||↓pain, ↓TNF-α, ↓prostaglandin (PGE2)||88|
|Male Sprague-Dawley rats||Ischemia-reperfusion injury (myocardial)||i.p.||Modest ↓infarct size, ↓TNF-α||200|
|C57BL/6J mice c||Aβ inflammation||i.p.||↓IL-1β, ↓iNOS||117|
|2.5 or 10 mg/kg|
|Male BALB/c mice||Ovalbumin (asthma)||i.p.||↓serum antibodies, ↓IL-2, IL-4, and IFN-γ||140b|
|Male ddY mice||Focal cerebral ischemia||i.p.||↓infarct size, ↓neutrophil MPO activity||129b|
|various times surrounding occlusion|
|Female NOD mice||Diabetes||i.p.||↓disease incidence, ↓IL-6 and IL-12, ↑IL-4 and IL-10||124b|
|5 injections per week for 4 weeks|
|Female B6C3F1 mice||sRBC||Oral||Modest ↓antibody production||55b|
|Male ICR mice||DNBS colitis||i.p.||↓inflammation, ↓colon weight:length ratio, ↓iNOS, IL-1β, ↑IL-10||95b|
|Male Wistar rats||None||i.p.||↓blood leukocytes and lymphocytes, ↓B, T and CTL cells, ↑NK and NKT cells||201|
|2.5 or 5 mg/kg|
|Male CD-1 mice||Diabetes||i.p. or i.n.||↓diabetic pain, ↓density of microglial cells||81b|
|0.1–2 mg/kg i.n.|
|1–20 mg/kg i.p.|
|Male C57BL/6 mice||Streptozotocin-induced diabetes||i.p.||↓disease, ↓TNF-α, NF-κB activity, ICAM-1, VCAM-1, iNOS, p-p38, p-JNK, ↑p-AKT||120b|
|Male Wistar rats||TNBS colitis||i.p.||Modest ↓disease, ↓colonic contractions, ↓neutrophil MPO activity||130|
|Male Wistar rats||Cecal ligation and puncture||i.p.||↑disease survival||184|
|once or up to 9 days|
|Female Sabra mice||Hepatic encephalopathy (bile duct ligation)||i.p.||Improved disease-associated cognitive impairments, ↓TNF-α||202|
|Male BALB/c mice||Ovalbumin (footpad)||i.p.||↓footpad swelling, ↓TNF-α and IFN-γ, ↑IL-10||188|
|Male Swiss OFI mice||LPS i.p.||i.p.||↓mast cell infiltration, macrophage activation marker, ↓TNF-α||96|
|Female C57BL/6 mice||Experimental autoimmune hepatitis||i.p.||↓hepatic inflammation, ↓IL-2, TNF-α, IFN-γ, IL-6, IL-17A, IL-12, MCP-1 (CCL-2), and eotaxin, ↑MDSCs||86b|
|Male C57BL/6 mice||Ischemia reperfusion injury (liver)||i.p.||↓hepatic inflammation, ↓MIP-1α, ICAM, MIP-2, TNF-α, NF-κB activity, ICAM-1, iNOS, p-p38, p-JNK||118|
|3 or 10 mg/kg|
|C57BL/6 mice c||LPS i.v.||i.v.||↓vasodilation, leukocyte margination, and extravasation, ↓COX-2, TNF-α, and iNOS||121|
|1 or 3 mg/kg|
|Male C57BL/6 mice||LPS-induced pulmonary inflammation||i.p.||↓BALF lymphocytes, macrophages, and neutrophils, ↓TNF-α, IL-6, MCP-1 (CCL-2), and MIP-2||125b|
|Male Wistar rats||Meningitis (Streptococcus pneumoniae)||i.p.||Improved disease-associated cognitive impairments, ↓TNF-α||203|
|once or up to 9 days|
|C57BL/6 mice c||Cerulein (pancreatitis)||i.p.||↓disease, ↓TNF-α and IL-6, ↓neutrophil MPO||128b|
|Newborn pigs c||Hypoxia-ischemic brain injury||i.v.||neuroprotection, ↓IL-1||59b|
|Male Wistar rats||Ovalbumin (asthma)||i.p.||↓TNF-α, IL-6, IL-4, IL-5, and IL-13||127b|
|Male C57BL/6 mice||LPS-induced pulmonary inflammation||i.p.||↓inflammation, ↓BALF lymphocytes, macrophages, and neutrophils, ↓TNF-α, IL-6, MCP-1 (CCL-2), and MIP-2||132|
|Female C57BL/6 mice||None||i.p.||↑MDSCs||136b|
|Female C57BL/6 mice||Malaria (Plasmodium berghei)||i.p.||↓IL-6 and TNF-α||204|
|Male Sprague Dawley rats||Freund’s Adjuvant (osteoarthritis)||Transdermal||↓inflammation, ↓TNF-α||205|
|Male ICR mice||DNBS Colitis||i.p. or oral d||↓colon weight:length ratio, ↓neutrophil MPO||131|
|5–30 mg/kg for i.p. 10–60 mg/kg oral|
|Female NOD mice||Type 1 diabetes||i.p.||↓disease||206|
|5 injections/week for 10 weeks|
|Male A/J mice||Experimental autoimmune myocarditis||i.p.||↓disease, ↓lymphocyte populations in heart, ↓IL-6, IFN-γ, IL-1β, and MCP-1 (CCL-2)||126b|
|Male Wistar rats||Middle cerebral artery occlusion||i.c.v.||↓infarct size||149|
|Male Wistar rats||Middle cerebral artery occlusion||i.c.v.||↓infarct size, ↓TNF-α||207|
|Male Wistar rats||Sodium monoiodoacetate (osteoarthritis)||Intra-arterial||↓pain, ↓rolling and adherent leukocytes, ↓joint nerve demyelination||83b|
|Female C57BL/6 mice||Alcoholic liver disease||i.p.||↓liver damage, ↓neutrophils, ↓TNF-α, MIP-1, IFN-γ, IL-1β, and MCP-1 (CCL-2)||185|
|5 or 10 mg/kg|
|Male and female dogs||Osteoarthritis||Oral e||↓pain||208|
|2 and 8 mg/kg|
|every 12 h for 4 weeks|
|Male Wistar rats||Ulcerative tongue lesion||i.p.||↓inflammation||209|
|5 or 10 mg/kg|
|3 or 7 days|
|Female C57BL/6 mice||Spinal cord contusion||i.p.||↓spinal cord CD4 T cells, ↓IL-23A, IL-23R, IFN-γ, CXCL9, CLCL11, NOS2, and IL-10||189|
|1 and 24 h after injury, on day 3, then twice/week up to 10 weeks|
|Male Sprague-Dawley rats||Carrageenan-induced inflammation||Oral||↓hyperalgesia||210|
|100 or 10,000 μg/kg|
|Male Swiss mice||Haloperidol-induced inflammation||i.p||↓IL-1β and TNF-α, ↑IL-10||97b|
|twice/day up to 21 days|
|Male BALB/c mice||Corneal inflammation||Topical (ophthalmic)||↓pain, ↓neutrophils||56b|
|3% or 5%|
|Male ICR mice||Ischemia-reperfusion injury (kidney)||i.p.||↓kidney injury, ↓TH17 cells, ↑Tregs and Treg17 cells||152b|
|Female C57BL/6 and BALB/c mice||Syngeneic or allogeneic bone marrow transplant||i.p.||↓lymphocyte recovery||57|
|every other day for 2 weeks|
|BALB/c mice||Ovalbumin (asthma)||i.p.||↓airway resistance; ↓IL-4, IL-5, IL-13, and eotaxin||60b|
|5 or 10 mg/kg|
|three times at time of ovalbumin challenge|
CBD, Cannabidiol; DNBS, dinitrobenzene sulfonic acid; iNOS, inducible nitric oxide synthase; i.n. intranasal; i.p., intraperitoneal; JNK, c-jun N-terminal kinase; LPS, lipopolysaccharide; MDSCs, myeloid-derived suppressor cells; MPO, myeloperoxidase; sRBC, sheep red blood cell; TNBS, 2,4,6-trinitrobenzene sulfonic acid; Treg, regulatory T cell.
CBD effects and mechanisms of immune suppression in innate cells
One of earliest effects reported with CBD was in human mononuclear cells, 111,112 in which TNF-α, IFN-γ, and IL-1α were all suppressed (0.01–20 μg/mL CBD or 0.03–64 μM CBD). Later studies focused on human monocytic cells revealed that CBD can induce apoptosis in either HL-60 (1–8 μg/mL CBD or 3.2–26 μM CBD) 113 or primary human monocytic cells (1–16 μM CBD). 114,115 Macrophages are also targets, although they have been studied more commonly in animal models. Peritoneal macrophages were used early on to demonstrate that CBD (3 μg/mL or 10 μM) targets nitric oxide, 116 and this has also been a well-studied target of suppression by CBD in many tissues and cell types. The mechanism by which CBD suppressed nitric oxide involves suppression of endothelial 87 or inducible nitric oxide synthase (iNOS) 58,95,117–121 in response to various inflammatory stimuli. iNOS is known to be regulated by the transcription factor nuclear factor-κB (NF-κB), 122 which is comprised of p65 and other proteins, and becomes active after degradation of the inhibitory protein, IκB. Decreased expression of iNOS by CBD correlated with stimulation of the inhibitory IκBα protein and inhibition of NF-κB p65 protein expression. 119,120 Using peritoneal macrophages from diabetic mice stimulated ex vivo with LPS revealed that macrophages isolated from CBD-treated mice did not produce as much TNF-α or IL-6 as macrophages isolated from vehicle-treated mice. 123,124 A direct effect of CBD decreasing macrophage numbers in the bronchoalveolar lavage fluid was shown following intranasal LPS administration to induce pulmonary inflammation. 125 There was also decreased expression of F4/80 (a marker of macrophages) mRNA expression by CBD in heart tissue in experimental autoimmune myocarditis. 126 Although this study identified CBD only affecting F4/80 mRNA expression as opposed to F4/80 cell surface staining, it does suggest a novel target (i.e., heart tissue) of CBD in a relatively understudied autoimmune model.
IL-6 is a proinflammatory cytokine produced by many cell types, predominantly innate cells. Many studies have shown that circulating IL-6 is readily inhibited by CBD in inflammatory models, including diabetes, 124 asthma, 127 pancreatitis, 128 and hepatitis. 86 CBD treatment in vivo resulted in lower IL-6 production in peritoneal macrophages stimulated ex vivo with LPS, 124 in the pancreas in acute pancreatitis, 128 and in bronchoalveolar lavage fluid in LPS-induced pulmonary inflammation. 125
There have been some reports that CBD alters neutrophil function. Compromised MPO activity by CBD has been studied in several tissues, including brain, 129 colon, 130,131 lung, 125,128,132 and pancreas. 128 Interestingly, in the pulmonary inflammation studies with LPS, neutrophil cell counts in the bronchoalveolar lavage fluid were also decreased by CBD compared to LPS. 125,132 Together, the results suggest that CBD’s mechanism for neutrophil suppression involves both decreased numbers of neutrophils and compromised MPO activity.
There are two recent studies focused on CpG stimulation of IFN-α production from human plasmacytoid dendritic cells. 133,134 While these studies are focused primarily on THC and other CB2 agonists, CBD was also used (1–10 μM) and did not affect IFN-α production. 133,134 It was interesting, however, that CBD suppressed the CD83 dendritic cell activation marker on dendritic cells derived from HIV + , but not healthy, individuals. 134 Reduction in dendritic cell CD83 signaling can compromise T cell function, 135 although additional studies using CBD in human dendritic cells and T cells are needed to establish the consequences of CBD-induced reduction in CD83 on HIV + dendritic cells.
Another mechanism by which CBD controls immune function is induction of regulatory cells. MDSC are innate, myeloid cells that possess the ability to control immune responses. Hegde et al. demonstrated that CBD induced CD11b + Gr-1 + MDSCs in the liver in a mouse hepatitis model. 86 Importantly, the isolated MDSCs were functional, that is, they suppressed proliferation of responder T cells ex vivo and improved liver function when administered before hepatitis induction. 86 CBD-induced MDSCs from the peritoneal cavity were able to attenuate inflammation in response to LPS. 136 In the experimental autoimmune encephalomyelitis (EAE) model, CBD induced MDSCs in the peritoneal cavity, but decreased the infiltration of MDSCs in the spinal cord and brain. 137 CBD-induced MDSCs from the peritoneal cavity were able to attenuate responder T cell proliferation ex vivo and attenuate EAE disease when administered in vivo. 137
CBD effects and mechanisms of immune suppression in lymphocytes
The area in which most of the effects of CBD in the immune system have been studied is T cells. Early studies examining rosette formation in response to sheep red blood cells (sRBCs) (generally considered to be a T cell response) revealed that CBD (1 and 100 μM) reduced this response. 138 Phytohemagglutinin (PHA)-stimulated IFN-γ production in T cells has also been shown to be inhibited by CBD (0.01–20 μg/mL or 0.03–64 μM). 111,112 Other studies have provided further evidence that T cell-produced IFN-γ is a critical target of CBD suppression. CBD inhibited IFN-γ production from lymph node cells isolated from arthritic mice stimulated ex vivo with collagen, 139 and from splenocytes isolated from NOD mice stimulated ex vivo with ConA. 123,124 IFN-γ production from splenocytes isolated from untreated mice was suppressed by CBD following ex vivo stimulation with phorbol 12-myristate 13-acetate/ionomycin (PMA/Io). 140 In the latter study, a 1-h exposure of CBD to the mice was meant to mimic the time for CBD distribution before receiving antigen sensitization with ovalbumin to induce asthma-like disease. 140 Thus, CBD’s ability to compromise various cytokines at the time of antigen sensitization might suggest that CBD affects primary activation of T cells, as has been suggested as part of the mechanism for other cannabinoids, such as THC. 141 Indeed, we have shown that a 30-min pre-treatment with CBD (0.1–20 μM) suppressed IFN-γ production in mouse splenocytes in response to PMA/Io or anti-CD3/CD28. 55 In these studies, it was shown that the mechanism by which CBD suppressed IFN-γ occurred at the level of transcription and that two important transcription factors for IFN-γ, activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT), were inhibited by CBD, suggesting a transcriptional mechanism for suppression. 55 CBD-induced suppression (0.1–10 μg/mL or 0.3–32 μM) of Ifng mRNA expression was shown using PHA-stimulated human PBMCs. 142 Given the many reports that IFN-γ seems to be a sensitive target of suppression by CBD, it was surprising that Ifng mRNA was not affected by CBD (5 μM) using encephalitogenic T cells stimulated by antigen-presenting cells (APCs) and myelin oligodendrocyte glycoprotein peptide (MOG35–55) in vitro. 143 However, CBD did inhibit expression of IFN-γ receptor 1 and CBD increased several IFN-γ-responsive genes known to attenuate T cell proliferation. 143 Overall, the data reveal that an important part of CBD’s action in the immune system is its ability to affect IFN-γ in multiple ways. Not only did CBD directly suppress IFN-γ production through a transcriptional mechanism under several conditions 55,142 but also suppressed IFN-γ receptor expression, and increased IFN-γ-induced genes that subsequently attenuate other immune targets. 143
A few other T cell-derived cytokines have been shown to be targets of CBD. As noted above, IL-6 is a critical target of CBD in many cells and tissues, 82,84,86,97,125–128,132 many of which are innate cells. However, IL-6 was also suppressed by CBD (5 μM) using encephalitogenic T cells stimulated by APCs and MOG35–55 in vitro, 144 and “IL-6 signaling” as a critical pathway suppressed by CBD. 143 Interestingly, “IL-17 signaling” was also identified as a critical pathway suppressed by CBD (5 μM) in T cells in vitro. 143 It should be noted that IL-6 promotes the differentiation of TH17 cells, 145 so the simultaneous suppression of IL-6 and IL-17A by CBD is consistent with CBD suppressing TH17 cell differentiation. Indeed, CBD (1–20 μg/mL or 3.2–64 μM) suppressed IL-17A production in human CD3 + T cells (derived from healthy patients or patients with MS or nonseminomatous germ cell tumors) stimulated ex vivo with PMA/Io. 146 Taken together with the data described in innate cells above, it is clear that CBD’s action in inflammation and immune function involves suppression of cytokine production from many different cell types.
The ability of CBD to suppress transcription factors such as NFAT, AP-1, and NF-κB likely accounts for its widespread suppression of many cytokines. 74,82,118–120,147–149 Some of the studies suggest that CBD increased, or perhaps stabilized, expression of IκB as part of the mechanism by which it suppresses NF-κB. 119,120,147 CBD (4 μM) stimulated IκB-α expression in high glucose-treated human coronary artery endothelial cells. 119 CBD induced expression of IκB-α in heart tissue from diabetic mice in vivo 120 and in LPS-stimulated microglial cells in vitro (CBD 1–10 μM). 147 It is interesting that NF-κB activity has not yet been identified as a target in T cells, suggesting that CBD-mediated suppression of NF-κB plays a bigger role in mediating anti-inflammatory effects in non-T cells.
Certainly, some of the dysregulation of these transcription factors is the result of suppression of various kinases upstream of their activation. Extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 MAPKs have all been identified as targets of suppression by CBD in various cell types. 74,80–82,118,120 Of these reports, one was conducted in human T cells. 80 In these studies, CBD (5 μM) was shown to suppress expression of total and phosphorylated p38 at the 16-h timepoint following CBD treatment. The authors also showed that the CBD-mediated inhibition of phosphorylated p38 was reversed by SR1445328 or tocopherol, suggesting that CBD acts through CB2 and that the mechanism of suppression involves reactive oxygen species (ROS) production. 80
Although well studied in cancer cell lines and primary tumor tissue, CBD-mediated apoptosis is also a contributor to the immune suppressive mechanism. Initially CBD-induced apoptosis in T cells was described in Jurkat and MOLT4 human T cells. 80 In the same study, McKallip et al. observed increased apoptosis of mouse lymphoma cells injected into, and then recovered from, the peritoneal cavity of mice that were treated with CBD. 80 Since then, there has been a series of studies characterizing the mechanisms by which CBD induced apoptosis in mouse immune cells. CBD (1–16 μM) was shown to induce apoptosis in mouse thymocytes and EL-4 T cells. 150 The same group demonstrated that CBD (1–16 μM) induced apoptosis in mouse splenocytes, including assessment of CBD-induced apoptosis by cell type (B220 + B cells and CD4 + and CD8 + T cells). 151 In both studies, CBD increased ROS, and CBD-mediated apoptosis was attenuated by N-acetylcysteine. 150,151 Wu et al. further demonstrated that the CBD increased ROS-activated caspase-8 to mediate apoptosis. 151 In follow-up studies in human monocytes, Wu et al. noted that CBD (1–16 μM) readily induced apoptosis, but that the effect of CBD on apoptosis was lost if the monocytes were pre-cultured for 72 h. 114 The authors suggest that the differential responsiveness to CBD was due to an increase in antioxidant capacity in cultured cells, which is a thought consistent with the mechanism by which CBD induced apoptosis in mouse lymphocytes. 150,151 CBD-induced apoptosis (1–16 μM CBD) in human monocytes was due to a cascade of intracellular events, including opening of the mitochondrial permeability transition pore, depolarization of the mitochondrial membrane potential, oxidation of a lipid in the mitochondrial inner membrane, and mitochondrial ROS generation, leading to cytochrome C release. 115 Thus, this latest study demonstrates a critical role of the mitochondria in CBD-induced apoptosis.
Another important mechanism by which CBD acts to control immune responses is through regulatory T cell (Treg) induction. In the ConA model of hepatitis, CBD modestly enhanced Tregs in the liver as quantified by CD4 + Foxp3 + cells. 86 A confirmation of in vivo induced Tregs by CBD was noted in an ischemia-reperfusion injury model in the kidney, in which CBD returned the disease-induced reduction in CD3 + Foxp3 + cells to baseline. 152 Interestingly, in the ischemia-reperfusion kidney model, CBD also induced “TReg17 cells,” which were defined as CD3 + Foxp3 + CCR6 + STAT3 + . 152 It has been suggested that Treg17 cells help control a TH17 response. In vitro, CBD (5 μM) induced a CD69 + LAG + population in CD4 + CD25 − cells, which were identified as one type of regulatory cell, and induced Il10 mRNA expression. 153 We showed in vitro that CBD (1–15 μM) induced functional CD4 + CD25 + Foxp3 + T cells under conditions of suboptimal stimulation and that Il10 mRNA expression was induced. 154
There are only a few studies in which B cells are identified as targets of CBD. CBD given at 25 mg/kg by intraperitoneal (i.p.) injection modestly reduced the sRBC-induced plaque-forming cells, which is a measure of antibody production. 155 We conducted a similar study using oral administration of CBD and also found modest inhibition of antibody production. 55 Other studies have shown that CBD robustly inhibited the sRBC-induced antibody production in vitro, 55 suppressed ovalbumin-induced IgM, IgG1, and IgG2a in an in vivo asthma model, 140 and reduced expression of activation markers such as major histocompatibility complex II, CD25, and CD69, on B cells. 153 CBD has also been shown to induce apoptosis in B cells. 151 Overall, the results suggest that B cells can be targets of suppression by CBD.
CBD-induced neuroprotection by suppression of microglial cell activation
There is no doubt that many of the mechanisms already identified for innate cells and lymphocytes also account for CBD’s ability to decrease microglial cell activation. CBD (1–16 μM) induced apoptosis in microglial cells, 156 which was dependent on activation of caspase 8 and 9, and was reversed in the presence of an agent that depletes cholesterol and disrupts lipid rafts. 156 These results suggest that CBD-induced apoptosis is dependent on lipid raft formation, 156 and indeed, this observation was confirmed by another group in BV-2 microglial cells. 157
BV-2 microglial cells have been used as a model in several articles, in which detailed transcriptional effects of CBD have been evaluated. 147,157–159 The mechanisms contributing to CBD (10 μM)-mediated suppression of LPS-stimulated cytokine production in microglial cells includes decreased activation of the Toll/IL-1 receptor domain-containing adapter-inducing IFN-β (TRIF)/IFN-β/signal transducer and activator of transcription (STAT) signaling pathway. 147 CBD suppressed LPS-stimulated NF-κB activation, and induced LPS-stimulated STAT3 activation, which has been shown to suppress NF-κB activation. 147 CBD (10 μM) was shown to affect several genes involved in lipid metabolism in unstimulated BV-2 cells, 157 which might account for CBD’s ability to increase anandamide 58,65–67,84,157,160 or could account for CBD’s dependence on lipid raft formation to induce apoptosis 156,157 Follow-up studies examining CBD’s effects in unstimulated BV-2 cells demonstrated that CBD (10 μM) alters zinc homeostasis, oxidative stress, and glutathione levels in microglial cells. 158,159 A recent study demonstrated that CBD alters microRNA (miRNA) expression, 161 and two of the CBD miRNA targets identified are discussed. First, CBD downregulated miR146-a, which acts as a negative regulator of inflammation, in both resting and LPS-stimulated cells, thereby contributing to CBD’s ability to downregulate proinflammatory cytokines. 161 Second, CBD upregulated miR-34a, which has several roles in cell survival, such as cell cycle, apoptosis, and differentiation. 161 These results show that CBD-induced alterations in miRNA expression are involved in the mechanism by which CBD suppresses immune function.
In vivo, CBD has been shown to decrease microglial accumulation in the spinal cord in diabetic mice, 81 which might contribute to attenuation of neuropathic pain, and CBD decreased haloperidol-induced activation of reactive microglial cells. 97 CBD’s suppression of TNF-α production from microglial cells in vitro was mediated by A2A adenosine receptors in EOC-20 mouse microglial cells (0.5–5 μm) 89 or rat retinal microglial cells (1 μM). 91
CBD Effects in Autoimmune Disease Models
EAE and MS
The immunosuppressive and neuroprotective mechanisms of CBD make it an ideal therapeutic candidate for MS, a neurodegenerative autoimmune disease of the CNS that affects ∼2.5 million people worldwide. The average age of onset is around 30 years, and symptoms can vary greatly for each patient based on the lesion locations within the CNS. 162 Two models frequently used in the laboratory environment to study MS are the EAE and Theiler’s murine encephalomyelitis virus (TMEV) models, and an increasing number of studies have shown promising results with CBD using these models ( Table 5 ). In 2011, Kozela et al. successfully demonstrated that CBD (5 mg/kg i.p.) administered at the onset of disease attenuated clinical disease, microglial activation, and T cell infiltration into the CNS in EAE, and that CBD reduced T cell proliferation in vitro. 163 CBD showed similar effects in the TMEV model, in which Mecha et al. demonstrated that CBD (5 mg/kg i.p.) administered for the first 10 days following disease onset reduced clinical disease and neuroinflammation by decreasing microglial activation and immune cell trafficking signals in the CNS. 164 Use of MOG35–55-specific T cells isolated from EAE mice in vitro has also been extremely vital to determining how CBD might be affecting T cells in these and other disease models. As outlined above, in the T cell section, in vitro CBD treatment of MOG35–55-specific T cells co-cultured with APCs with CBD suppressed IL-17A and IL-6 production, suggesting CBD suppressed TH17 development; however, production of Il10 mRNA was potentiated with CBD treatment, suggesting that CBD may have multiple suppressive mechanisms. 144 In vitro treatment of MOG35–55-specific T cells with CBD induced a Treg with a CD4 + CD25 − LAG3 + CD69 + phenotype, promoted upregulation of anergy-associated genes, such as Lag3, Erg2, and Il10, and altered the balance between STAT3 and STAT5 activation. 153 In another study, CBD administered during disease onset increased the number of functional MDSCs present within the peritoneal cavity, decreased neuroinflammation, and reduced IL-17A and IFN-γ in the serum. 137 When splenocytes from these mice were restimulated ex vivo, the CBD-treated mice had significantly decreased levels of IL-17A and IFN-γ, and increased levels of IL-10 in the supernatants. 137 Finally, a recent study using an adoptive transfer EAE model showed a reduction in neuroinflammation, demyelination, and axonal damage with CBD treatment during disease onset. 165 Adoptive transfer EAE is a variation of the EAE model induced by transfer of encephalitogenic T cells into naive mice, which allows experiments performed with this model to focus more on the T cell-specific mechanisms of pathogenesis in the EAE model. From the accumulation of data, it is obvious that multiple immune cell types, proinflammatory and anti-inflammatory, within the EAE model are modulated by CBD, but overall, CBD appears to downregulate proinflammatory pathways and upregulate anti-inflammatory pathways in the EAE model.
Cannabidiol Effects in Experimental Autoimmune Encephalomyelitis
|EAE in ABH||In vivo||In vivo: 0.5–25 mg/kg i.p.||No effects||211|
|EAE in C57BL/6||In vivo and in vitro||In vivo: 5 mg/kg i.p. in vitro: 1, 5, and 10 μM||in vivo: ↓disease severity, ↓T cell infiltration into the CNS, ↓microglial activation, ↓axonal damage in vitro: ↓T cell proliferation||163a|
|TMEV in SJL/J||In vivo and in vitro||In vivo: 5 mg/kg i.p. in vitro: 1 and 5 μM||in vivo:↓disease severity, ↓leukocyte infiltration into the CNS, ↓microglial activation, ↓CCL2 (MCP-1), ↓CCL5, ↓IL-1β ↓TNF-α in vitro: ↓sVCAM-1 production from endothelial cells, ↓leukocyte adhesion, ↓CCL2 (MCP-1)||164a|
|MOG35–55-specific T cells from EAE mice||In vitro||In vitro: 0.1, 1, and 5 μM||in vitro: ↓IL-17A, ↓IL-6, ↑IL-10||144a|
|MOG35–55-specific T cells from EAE mice||In vitro||In vitro: 5 μM||in vitro:↓IL-17A, ↓IL-6, ↑IL-10, ↑EGR2, ↑CD4 + CD25 − CD69 + LAG3 + phenotype, ↑STAT5/↓STAT3, ↓B cell activity, ↑Nfatc1, ↑Casp4, ↑Cdkn1a, ↑Icos, ↑Fas||153a|
|EAE in C57BL/6||In vivo||In vivo: 5 mg/kg i.p.||in vivo: ↓disease severity, ↓leukocyte invasion, ↓demyelination, ↓TNF-α, ↓IFN-γ, ↓IL-17A||212|
|EAE in C57BL/6||In vivo||In vivo: 10 mg/kg i.p.||in vivo: ↓disease severity, ↓FAS ligand, ↓ERK phosphorylation, ↓Caspase-3 activity, ↓Bax/↑Bcl-2, ↓p53-p21 activation, ↓apobody formation||166a|
|MOG35–55-specific T cells from EAE mice||In vitro||In vitro: 5 μM||in vitro: ↓IL-1β, ↓IL-3, ↓Xcl1 mRNA, ↓IL-12a mRNA, ↑Dusp6 mRNA, ↑Btla mRNA, ↑Lag3 mRNA, ↑Irf4 mRNA, ↑IL-10 mRNA||143a,b|
|EAE in C57BL/6||In vivo||In vivo: 10 mg/kg i.p.||in vivo: ↓disease severity, ↓leukocyte infiltration, ↑PI3k/Akt/mTOR phosphorylation, ↑S6k phosphorylation, ↑BDNF expression, ↑PPAR-γ, ↓IFN-γ, ↓IL-17A, ↓JNK activity, ↓p38 MAP kinase activity||167a|
|Adoptive Transfer EAE in C57BL/6||In vivo and in vitro||In vivo: 5–50 mg/kg i.p in vitro: 1, 5 & 10 μM||in vivo: ↓disease severity, ↓leukocyte invasion, ↓demyelination, ↓axonal damage, ↓microglial activation, ↓CB2 receptor expression in CNS, ↓GPR55 receptor expression in CNS in vitro: ↓Cell viability, ↓IL-6, ↑apoptosis, ↑ROS||165a|
|EAE in C57BL/6||In vivo||In vivo: 20 mg/kg i.p||in vivo: ↓disease severity, ↓leukocyte invasion, ↓IL-17A, ↓IFN-γ, ↓RORγT, ↓T-bet, ↑IL-10, ↑MDSC ex vivo: ↓IL-17A, ↓IFN-γ, ↑IL-10||137a|
CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; ERK, extracellular signal-regulated kinase; STAT, signal transducer and activator of transcription; TMEV, Theiler’s murine encephalomyelitis virus.
In addition to its immunomodulatory effects, CBD’s neuroprotective properties in the EAE model also indicate its therapeutic potential in MS. CBD has been shown to decrease the activation of proapoptotic proteins, such as caspase-3 and Bax, 166 and to counteract the effects of EAE on the PI3K/Akt/mTOR pathway, JNK, and p38 MAP kinases in the CNS of EAE mice. 167 Interestingly, the study from Giacoppo et al. found the PI3k/Akt/mTOR pathway was upregulated in neural tissues when EAE mice were treated with CBD. 167 However, Kozela et al. 153 observed a reduction in the activation of Akt in vitro in MOG35–55-reactive T cells, which might suggest a differential role for CBD’s effects on the PI3K/Akt/mTOR pathway in various cell types.
Despite the growing number of studies involving the neuroprotective and immunosuppressive effects of CBD, the majority of human studies involving cannabinoids and MS have been focused on the use of THC:CBD mixtures, with a particular focus on Sativex. Clinical studies that have been performed have shown that Sativex has beneficial effects on spasticity, mobility, bladder function, and pain in MS patients, and is well tolerated 22,25,28,31,168–175 ; however, there has been little focus on the neuroprotective and immunosuppressive effects of THC:CBD mixtures in MS, and so it is difficult to say at this point if the successful results observed with CBD in the animal models of MS will be observed in MS patients. For a more complete review on the effects of Sativex in MS, see Zettl et al. 176
Other autoimmune disease states
CBD has been shown to attenuate experimental autoimmune hepatitis, 86 experimental autoimmune myocarditis, 126 and autoimmune diabetes 123,124 in mice. There are few studies done with CBD only in human autoimmune diseases. In human patients, CBD at 20 mg/kg did not reduce clinical Crohn’s disease. 177 However, CBD is effective at attenuating intestinal inflammation in other models of human inflammatory bowel disease, 82,96 so it is possible that CBD could be effective at higher doses. Indeed, CBD as Epidolex for epilepsy in children is being used as high as 20 mg/kg, but CBD doses as high as 300 mg/kg have been evaluated, and have not exhibited significant adverse effects. 178
CBD Immune Enhancement Effects
Much of the data support the fact that CBD is immune suppressive and anti-inflammatory; however, there have been a few reports over the years that CBD has produced some immune enhancing effects ( Table 6 ). The potential for CBD, and other cannabinoids, to produce immune enhancing effects has been attributed to differences in hormetic (i.e., biphasic) responses depending on CBD concentration/dose, cell culture conditions, including serum presence and/or percent, immune stimulant, and magnitude of cellular activation in response to the immune stimulant. Indeed, studies from our laboratory and others have shown that CBD either enhanced or suppressed cytokine production (IL-2 and IFN-γ) in response to relatively low or high degree of immune stimulation, respectively. 154,179,180 The mechanism for the differential responsiveness likely involves alterations in intracellular calcium, as CBD increases intracellular calcium in mouse splenocytes regardless of the increase of intracellular calcium produced by the immune stimulant. 179 In addition, the differential cytokine production was correlated with nuclear expression of the NFAT transcription factor, 179 which is calcium responsive. Interestingly, CBD’s ability to increase intracellular calcium also likely accounts for some of the other enhancing effects, including stimulation of neutrophil degranulation, 181 chemotaxis, 182 and mast cell/basophil activation. 183