As a member of the immune system, mast cells have a direct effect on inflammation and disease tied to inflammation (just about everything these days).
See why this system may be the linchpin?
Mast cell activation and mental health
Why are most types of allergic diseases increasing over the past few decades?
These symptoms (Table 1) originate from mediators secreted (released) from mast cells and include flushing, pruritus, hypotension, gastrointestinal complaints, headaches, irritability, malaise, memory loss, and neuropsychiatric issues.
THC, on the other hand, mimics anandamide but lingers for too long and only works in one direction (up up up for CB1 activity).
Flow cytometric analysis of MDSC from peritoneum 12 h after injecting with vehicle or CBD in WT or Trpv1 −/− (VR1-KO) mice. Frequencies (A) and absolute numbers of MDSC (B) are depicted. Data represent mean ± SD (n=3 mice). Flow cytometric analysis of MDSC from peritoneum 12 h after injecting with vehicle or CBD in WT mice with or without pretreatment of mice using specific inhibitor (BADGE) to block PPARγ receptors (C, D). Data represent Mean ± SD (n=4 mice). Student’s t-test: ***P<0.001. E) Murine mast cells secrete G-CSF in response to CBD in a PPARγ-dependent manner. Normal murine cloned mast cells (MC/9) were treated in culture (10 6 cells/well) with 1 or 10 μM CBD. In some wells PPARγ-inhibitor BADGE was added at 1 or 10 μM as indicated. Culture supernatants were harvested after 24 h and analyzed for G-CSF by ELISA. Data represent mean ± SD of triplicate determinations and representative of two experiments. Student’s t-test: **P<0.01, *P<0.05.
Although, a partial or weak agonist for the vanilloid receptors (EC50, 3.5 μM), CBD has been shown to function by activating Trpv1 in several models of inflammation (19, 53). In this study, using Trpv1 −/− mice we have observed that induction of MDSC by CBD in the naïve peritoneum was completely independent of Trpv1. This is particularly interesting because anti-inflammatory effect of CBD in experimental hepatitis was Trpv1-dependent and was associated with increased MDSC numbers in liver (27). This suggests that unlike local, robust MDSC response to CBD in the peritoneum in naïve mice, migration and accumulation of MDSC in other organs, especially during active inflammatory response may involve Trpv1-dependent mechanisms. Additionally, different mechanisms may come into play in activated versus normal conditions, and Trpv1 may have different roles during inflammation as opposed to naïve system. Further, peritoneum being recognized as a unique immune organ, it is likely that phenotype of peritoneal mast cells, with respect to expression and/or function of PPARγ, release chemokine mediators such as G-CSF and their involvement in the induction of MDSC in response to CBD may be organ-specific. It is well known that CBD has a complex pharmacology (1) and functions by binding and activating different receptors in different models. Some in vivo effects of cannabidiol have been previously attributed to CB1 (26, 54–56) and CB2 receptors (55, 57, 58), although the general agreement in the field is that CBD exhibits little affinity towards these cannabinoid receptors. Recently, CBD has been shown to function by binding to adenosine receptor A2A (46, 48, 57). Neuroprotective effect of CBD in hypoxic-ischemic brain damage (57), anti-inflammatory effect in retina (48) and acute lung injury (46) have been shown to involve A2A receptors. We studied the possible involvement of these receptors initially in the current study by pre-injecting CB1/ CB2 antagonists or specific A2A inhibitor. We did not see any effect of these inhibitors on the induction of MDSC by CBD in vivo (data not shown).
In the current study, therefore, we investigated if administration of CBD into normal mice would induce MDSC. Interestingly, we found that CBD caused robust induction of immunosuppressive CD11b + Gr-1 + MDSC in naïve mice which was associated with significant upregulation of G-CSF, M-CSF and CXCL1. We demonstrate that this response is dependent on mast cells, and primarily mediated by PPARγ.
WT mice (n=4) were injected with vehicle or different doses of c-48/80 i.p. and peritoneal exudate cells were analyzed by flow cytometry for MDSC. Representative dot plots with frequency of gated CD11b + Gr-1 + MDSC are shown (A). Absolute MDSC numbers from 4 mice are represented as mean ± SD (B). C, D) Flow cytometric analysis for MDSC subtypes as described before. Student’s t test, **P<0.01 compared to vehicle control.
Blocking of G-CSF inhibits MDSC induction in vivo
Adoptive transfer of mast cells in mast cell-deficient mice
Cell lysates (20μg protein per lane) were separated by SDS-PAGE and electrophoretically transferred onto nitrocellulose membrane using a semi-dry transfer unit (Bio-Rad) followed by blocking with 5% blotting grade dry milk in tris-buffered saline containing 0.1% tween-20. Next, membranes were incubated with anti-mouse-arginase (1:2000), washed and probed with anti-mouse HRP secondary antibody (1:15000). The blots were developed using ECL reagent (GE) on to Kodak BioMax chemiluminescence film. β-actin was used as the internal control.
Cannabidiol, SR141716A (SR1, CB1 antagonist) and SR144528 (SR2, CB2 antagonist) were provided by National Institute of Drug Abuse. The monoclonal antibodies (mAbs), FITC-conjugated anti-CD11b (clone: M1/70), anti-Ly6C (HK1.4), PE-conjugated anti-Gr-1 (anti-Ly6G/Ly6C, clone: RB6-8C5), anti-Ly6G (clone: IA8), anti-CD3, anti-CD4, anti-CD8, anti-CD31, anti-CD11c, anti-F/480, anti-Ki-67, Alexa 647-conjugated anti-CD11b and purified anti-CD16/CD32 (mouse Fc receptor block) were from Biolegend (San Diego, CA). The anti-arginase Ab was obtained from BD Transduction Laboratories. The anti-Gr-1 microbeads, magnetic sorting columns and equipment were from Miltenyi Biotech. Adenosine (A2A) receptor antagonist 4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), PPARγ antagonist 2,2-Bis[4-(2,3-epoxypropoxy)phenyl]propane (Bisphenol A diglycidyl ether or BADGE) and PPARγ agonist 5-[[4-[(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-2,4-thiazolidinedione (troglitazone) were purchased from Tocris Bioscience. Cell culture grade concanavalin A, L-arginine, L-ornithine standard, Ninhydrin reagent, red blood cell lysis buffer and all other chemicals and reagents were from Sigma-Aldrich (St. Louis, MO).
Mast cell activator (C-48/80) induces accumulation of MDSC
The potent anti-inflammatory and immunomodulatory effects of cannabidiol has been demonstrated in various pre-clinical disease models such as murine collagen induced arthritis (16), high glucose-induced endothelial cell inflammatory response and barrier disruption (17), β-amyloid induced neuroinflammation (18), acute carrageenan-induced inflammation (19), development of type I diabetes in NOD mice (20), hepatic ischemia/reperfusion injury (21), LPS-induced inflammation in brain (22) and MS like disease (23). In line with its wide spectrum of action, CBD has been shown to bind to various receptors such as vanilloid receptor (Trpv1), cannabinoid receptors (CB1 and CB2), Adenosine receptor 2A (A2A), α-1 and α-1-β glycine receptors (18) with varying affinities, and has been shown to function via different receptors in different models. Recent studies demonstrated that CBD directly activates peroxisome proliferator-activated receptor PPARγ, a non-cannabinoid nuclear receptor, to influence gene expression (24–26) and exert its effects. Although, CBD is shown to decrease T cell responses and inhibit inflammatory cytokine production in these models, little is known about the effect of CBD on important suppressor cell populations. Recently, we showed that CBD was able to ameliorate T cell-mediated acute liver inflammation in ConA-induced as well as D-Galactosamine/Staphylococcal Enterotoxin B (D-Gal/SEB)-induced hepatitis in mice, which was associated with significant increase in MDSC in livers (27). Because inflammation is also known to trigger MDSC, it was not clear from these studies if CBD further augmented the inflammation-driven MDSC induction.
CBD (20 mg/kg) was injected i.p. into groups of WT mice (n=4) for each time point. G-CSF, GM-CSF, CXCL1 and M-CSF levels in the peritoneal exudates were analyzed by ELISA (A-C). Blocking experiment with anti-G-CSF in vivo: WT mice (n=3) were injected with isotype control IgG or anti-G-CSF Ab (10μg/mouse) 1 h before injecting with CBD (20mg/kg). Peritoneal exudate cells were harvested after 12 h, and analyzed by FACS for MDSC. Representative dot plots are shown for each treatment (D); Absolute number of MDSC per peritoneum (n=3 mice) (E). F) G-CSF levels determined by ELISA in the peritoneal exudates of WT and mast cell deficient Kit W/W-v mice 16 h after injection with 20 mg/kg CBD. Error bars indicate SD. Student’s t-tests: **P<0.01, *P<0.05.