The plant cannabis sativa, produces a variety of biologically active cannabinoids and related compounds. ∆⁹-tetrahydrocannabinol (THC) is the predominant molecule with 1–10% by weight. It was first extracted in 1964 by Gaoni and Mechoulum and is now known to be the primary molecule responsible for most of the biological effects of the cannabis plant [1]. Thereafter, in 1992, N-arachidonylethanolamine “anandamide” (AEA) was isolated from swine brains, which was the first endogenous cannabinoid related substance isolated from a mammal [2]. Related substances were also isolated from gastrointestinal tissues [3,4]. In the following years, multiple endogenous cannabinoid molecules like 2-arachidonylglycerol (2-AG), noladin ether, virodhamine, and oleoyl ethanolamine were identified and these substances were then named as “endocannabinoids”, which are derivatives of arachidonic acid conjugated with ethanolamine or glycerol. Endocannabinoids, their receptors, and metabolic pathways form the “Endocannabinoid System”, a term which was first used by Di Marzo and Fontana [5].

Among the wide variety of endocannabinoids, AEA and 2-AG have attracted a significant number of researchers in the endocannabinoid area. AEA is synthesized by the enzymes N-acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD), α/β-hydrolase-4 (Abh4), and phospholipase-C (PLC)-catalyzed cleavage of NAPE to phosphoanandamide and then to AEA [6,7]. It is mainly catabolized by fatty acid amide hydrolase (FAAH), the enzyme which also catabolizes the non-cannabinoid fatty acids [8]. In addition to FAAH, N-acylethanolamine acid amidase (NAAA) has also been identified as another hydrolase for AEA [9]. 2-AG is synthesized by phospholipase C (PLC) and diacyl-glycerol-lipase (DAGL), and catabolized mainly by monoacylglycerol-lipase (MAGL) [10]. Although MAGL is the predominant enzyme for 2-AG metabolism, α/β-hydrolase-6 (Abh6) and α/β-hydrolase-12 (Abh12) also contribute to a minor degree. FAAH has a negligible effect in 2-AG catabolism [7]. In macrophages, it was also shown that carboxylesterase-1 (CES1) and palmitoyl protein thioesterase-1 (PPT1) could hydrolyze 2-AG [11,12]. Nevertheless, cyclooxygenase-2 (COX-2) is involved in the oxidation of both 2-AG and AEA [13]. COX-2 oxidizes AEA to generate prostamides, like prostaglandin H₂ ethanolamide (PGH₂-EA) and prostaglandin H₂ (PGH₂), respectively [14], and 2-AG to prostaglandin H₂ glycerol (PGH₂-G) [14,15].

Cannabinoids exert their biological effects mainly through two 7-transmembrane (TM) receptors, namely cannabinoid receptor CB₁ and CB₂ [16]. However, they can also bind to other targets like transient receptor potential vanilloid receptor1 (TRPV1) [7,17], orphan receptors G protein-coupled receptor-55 (GPR55) [18,19], GPR18 [20], GPR110 [21], GPR119 [22], and peroxisome proliferator-activated receptors (PPARs) (mostly PPARα) [22]. The cannabinoid receptors are G-protein coupled receptors (GPCRs) (G_i/o) and human CB₁/CB₂ receptors share 44% overall homology [16,23]. Since mice have been used in several cannabinoid related studies, it is also important to note that human and mouse CB₁ receptors share 96% [24], and CB₂ receptors share 82% homology [25], where mouse CB₁ and CB₂ receptors share 66% homology [25]. Both CB₁ and CB₂ receptors are negatively coupled to adenylyl cyclase and stimulate mitogen-activated protein kinase (MAPK). Furthermore, they also activate K⁺ channels and inhibit Ca⁺⁺ channels, both of which result in the inhibition of transmitter release via the activation of G_β/γ subunit [26,27,28].

CB₁ receptors are mainly located in the nervous system, nerve terminals and a wide range of tissues including adipose tissue, liver, gastrointestinal tract, whereas CB₂ receptors are expressed in peripheral tissues, primarily in immune cells, immune-related organs and tissues like tonsils, spleen, thymus and bone marrow [7,29,30]. However, similar to CB₁, CB₂ receptor expression has also been demonstrated in various tissues and cells such as the brain, spinal cord [31,32], lung, testes [23,33], osteoblasts, osteocytes, and osteoclasts [34].

Although most of the research has been focused on the central nervous system effects of cannabinoids, they have various biological effects in the immune, cardiovascular, gastrointestinal, and respiratory system [35]. These effects had attracted the interest in the clinical use of cannabinoids. However, most of the cannabinoid related drugs have been withdrawn from the market due to unacceptable central side effects, which are mainly mediated by CB₁ receptors. Today “Sativex^®” is the only cannabinoid-containing product, which consists of ∆⁹-tetrahydrocannabinol (THC) and cannabidiol, and have been approved for the spasticity of multiple sclerosis.

Cannabinoids have a significant contribution to the immune modulation [36], which suggests their importance in pathophysiological conditions and inflammatory diseases. The alteration of endocannabinoid levels by inflammation was observed in several studies performed in the brain, liver, coronary artery, and colon [37,38,39,40,41]. Furthermore, endocannabinoids and activation of the cannabinoid system have also been demonstrated to inhibit inflammatory responses in hepatitis [42], pulmonary inflammation [43], inflammatory pain [44], sepsis [45] and colitis [46] by reducing the recruitment of inflammatory cells and increasing the production of anti-inflammatory cytokines. Earlier studies have indicated that cannabinoids can inhibit antibody production [47,48]. THC has been shown to suppress Th1 and enhance Th2 cytokines, and AEA has been demonstrated to decrease T and B cell proliferation [49,50]. However, the immune-modulatory effects of cannabinoids can also lead to the down-regulation of immune responses, which can cause the augmentation of infections and tumorigenesis [51,52,53].

The intense involvement of cannabinoids in immunomodulation also suggested the role of CB₂ receptors as they are highly expressed in the immune system. CB₂ receptors are involved in B-cell differentiation [54], macrophage migration [55], and antigen processing [56]. CB₂ receptors also contribute to mast cell activation by generating nitric oxide (NO) and prostaglandin-E₂ (PGE₂) [57]. Besides CB₂ receptors, the existence of functional CB₁ receptors was also shown in mast cells [58], suggesting both receptors are also important in allergic diseases [59,60]. On the other hand, when used in higher concentrations, the effects of cannabinoids on inflammation can occur through cannabinoid receptor-independent mechanisms such as PPARs [61,62].

It is known that marijuana smoking can cause cellular damage in the lungs. However, there are some differences in the airways of marijuana and tobacco smokers [63,64]. Gong et al. have shown that both marijuana and tobacco smokers have goblet and basal cell hyperplasia with a tendency of higher hyperplasia in the marijuana group [65]. There was also cellular disorganization in more than 50% of marijuana smokers. In a study performed in primates, smoking marijuana was shown to induce bronchiolitis, alveolar cell hyperplasia, and fibrosis in greater incidence when compared to that of the cigarette group [66]. Therefore, it can be concluded that the plant-derived cannabinoids may have significant effects in the airways.

The synthesis of endocannabinoids in the airways is established in various cell types (Figure 1). Although the data about the expression of CB₁ and CB₂ cannabinoid receptors in human airways are not clear, it is known that they are mostly expressed by the immune cells within the airways [67,68]. CB₂ receptors are densely expressed on eosinophils [69,70,71] and monocytes, both of which also express CB₁ receptors [33,72,73,74]. Eosinophil recruitment to the airways is an important process in the chronic inflammatory state of allergic asthma. The significant amount of CB₂ receptors in human eosinophils indicates a critical response capacity of these cells to cannabinoids [75]. In this regard, 2-AG was shown to be a chemoattractant factor for human primary eosinophils [69,76,77]. Moreover, interleukin-5 (IL-5), which is an important mediator for eosinophil differentiation and priming, can enhance the effect of 2-AG [77]. Selective stimulation of CB₂ receptors with synthetic ligands is also able to modify the activity of eosinophils to chemoattractants [78]. These studies indicate that endocannabinoids have a significant contribution to eosinophil recruitment. Human monocyte-derived dendritic cells (DCs) isolated from peripheral blood and murine bone marrow-derived DCs express both CB₁ and CB₂ receptors, which can synthesize AEA and 2-AG [49,79,80]. Studies have shown that the activation of DCs by cannabinoid ligands can inhibit the release of inflammatory cytokines [49] and can suppress the immune response by inducing apoptosis of these cells [79]. The interaction of DCs with T cells should also be considered for the effects of cannabinoids in the immune response. Do et al. suggested that inflammation-induced 2-AG production by DCs can affect cannabinoid receptors on T cells and switch the immune response from Th2 to Th1, and T cells can produce endocannabinoids which may affect their receptors on DCs. [80]. The expression of cannabinoid receptors and their function is more complicated in neutrophils. Detectable CB₂ mRNA levels were shown in polymorphonuclear neutrophil cells [33]. However, Chouinard et al. have demonstrated that human neutrophils do not express a significant amount of CB₂ receptor protein, although they are responsive to selective agonists [70,71,81].

Several pathological conditions have been associated with a change in the expression of cannabinoid receptors, altered endocannabinoid tissue concentrations, or a change in their metabolism. Therefore, different therapeutic strategies were considered by modifying the endocannabinoid system, such as targeting CB₁/CB₂ receptors or interfering with their metabolism [6].

Due to the immunomodulatory effect of cannabinoids, a significant variety of studies were focused on their possible therapeutic potential on inflammatory diseases like asthma [43,67,68,92,93,104,109,110,111,112,113,114]. The molecular mechanisms mediating the effects of cannabinoids in allergic airway responses mainly depend on their effects on immune cells and the related release of cytokines [115]. In mice, treatment with the plant-derived cannabinoids, cannabinol and THC, was able to inhibit the expression of critical T cell cytokines and inflammatory response in ovalbumin-induced experimental allergic airway inflammation [116]. Using the ovalbumin model, Braun et al., have demonstrated that THC can inhibit cell proliferation, suppress cytokine and chemokine production, and stimulate regulatory T cells [112]. However, they have also suggested that these effects are probably mediated by cannabinoid receptor-independent mechanisms [112].

Giannini et al. demonstrated that the non-selective cannabinoid receptor agonist CP-55,940 can prevent allergen-induced bronchospasm, and reduce cough and leukocyte recruitment in the lung [67]. This effect could be blocked by both CB₁ and CB₂ receptor blockade, suggesting that both receptors were involved in this effect [67]. In guinea pig airways, the synthetic cannabinoid ligand WIN-55212-2 has demonstrated a reduction in airway neurogenic inflammation in vivo [91]. This was due to an inhibition of C-fiber nerve activity and mediated by the activation of CB₂ receptors. WIN-55,212-2 was also shown to inhibit airway plasma extravasation and bronchoconstriction induced by intra-esophageal HCl instillation in guinea-pigs, an effect which was also mediated through CB₂ activation [113]. CB₂ receptor modulation of airway sensory nerve activity and related cough reflex have been highlighted in several different studies [110,117]. These findings suggest that CB₂ receptors can contribute to both neurogenic airway inflammation and hyperreactivity as well. This may also be related to a species difference since studies underscoring the CB₂ receptor-mediated reactivity changes in the airways are mostly performed in guinea pigs. However, the crucial role of CB₂ activation in the regulation of pulmonary natural killer (NK) cell function has also been demonstrated in mice [118]. Therefore, blocking the CB₂ receptors in allergic airway inflammation may modulate NK cell response during airway inflammation. The plant-derived cannabinoid, cannabidiol, was also shown to inhibit allergen-induced contraction of airway smooth muscle, and reduce antigen-induced airway obstruction in guinea pigs [104]. This effect seems to be partly mediated by the inhibition of mast cell degranulation.

In the experimental studies performed in mice, selective CB₁ receptor agonists have been shown to inhibit inflammation-induced hyperreactivity in an in vitro model of nerve growth factor (NGF)-induced neurogenic inflammation [93]. The inhibitory effect of CB₁ receptors was demonstrated using two different synthetic selective CB₁ agonists ACEA and ACPA. In the same study, these selective ligands were also able to inhibit nerve mediated cholinergic contractions, further confirming the inhibitory role of CB₁ receptors in airway reactivity [93]. Moreover, in a model of experimental non-atopic asthma in mice, in vivo intranasal treatment with the CB₁ selective agonist, ACEA was shown to prevent airway hyperreactivity [111].

Inhaled AEA pre-treatment of guinea pigs has shown to prevent leukotriene-D₄ aerosol-induced bronchospasm [114], an effect suggesting the role of endocannabinoid targeted therapy in airway diseases. AEA has also been shown to reduce trans-epithelial resistance in airway cells, which indicates an increase in barrier permeability [119]. However, this effect seems to be mediated by the metabolism of AEA to one or more LOX and COX metabolites rather than CB₁ and CB₂ receptor-dependent mechanisms. Increased AEA levels in asthmatic patients may contribute to increased permeability of the epithelium through degradation to arachidonic acid metabolites. Therefore, preventing AEA hydrolysis in the airways may help to prevent epithelial permeability in asthma [119]. A strategy for this and to increase AEA tissue levels is to inhibit the activity of the FAAH enzyme, which is responsible for the hydrolysis of AEA and other related amidated signaling lipids, such as PEA, N-oleoylethanolamide (OEA) and linoleoylethanolamide (LEA). Inhibition of FAAH has been shown to produce antitussive effects in guinea pigs [120]. This effect was suggested to be mediated by elevated fatty amino acids, which act on cannabinoid (CB₂) receptors on vagal sensory nerves [120]. Therefore, increasing the endocannabinoid levels by FAAH inhibition can be a promising strategy as a new treatment option for antitussive therapy.

The effect of endocannabinoids was studied in the LPS-induced experimental acute lung injury (ALI) model in mice [121]. It is well known that ALI may occur due to sepsis, pneumonia, acid aspiration, toxic inhalation, etc. In their study, Costola-de-Souza et al. showed that treatment with the MAGL inhibitor, JZL184 attenuated the pathological changes of ALI by increasing 2-AG levels in the lungs. However, in their study, neither CB₁ nor CB₂ receptor antagonists were able to fully block the effect of JZL184, suggesting the involvement of other mechanisms such as stimulation of non-cannabinoid receptors or an increase in non-cannabinoid products like prostaglandins. Abohalaka et al. studied the effects of FAAH or MAGL inhibitor treatments in LPS-induced airway inflammation and airway hyperreactivity [109]. These inhibitors were applied either systemically (i.p. route) or locally (i.n.) before LPS administration to mice. Both FAAH inhibitor and MAGL inhibitor treatments were found to be effective in preventing airway hyperreactivity, whether they were applied via the i.p. or i.n. route. The treatments were also able to prevent the histopathological changes in the lungs, except the local i.n. application of the FAAH inhibitor URB597 [109]. These effects were possibly mediated by the increased AEA and 2-AG levels in the lungs (unpublished data). These data suggest that increasing the concentration of endocannabinoids in the airways by the inhibition of primary endocannabinoid degrading enzymes FAAH and MAGL can prevent airway hyperreactivity and airway inflammation. However, more studies should be performed in order to clarify the effects of FAAH and MAGL inhibitors in the airways since different fatty amino acids are also substrates for these enzymes.

The endocannabinoid system also contributes to pulmonary fibrosis, which is a life-threatening disease. Endocannabinoids are shown to promote the progression of fibrosis in liver [122,123,124], kidney [125,126], heart [127], and skin [128]. Studies in mice suggest that CB₁ receptors are associated with radiation-induced pulmonary fibrosis [129]. Cinar et al. performed a detailed study in order to identify the contribution of CB₁ receptors in lung fibrosis, and have shown that both the genetic deletion of CB₁ receptors or their pharmacological inhibition with a CB₁ antagonist can attenuate lung inflammation and fibrosis, and hence increase animal survival in a mouse model of radiation-induced pulmonary fibrosis [130]. This finding indicates a different pattern than other studies in which cannabinoid CB₁ receptor stimulation has been raised as a therapeutic approach, as discussed above. This controversy may be due to the models used to induce fibrosis and related pathophysiology of fibrosis, which is different from acute or allergic airway inflammation. However, although CB₁ activation can prevent airway hyperreactivity and inflammation, CB₂ receptor antagonism has also been suggested as a therapeutic strategy for allergic diseases [78,118].

The studies about the contribution of the endocannabinoid system in the airways indicate the importance of both CB₁ and CB₂ receptors (Table 1). Among these two receptors, CB₁ subtype is more likely to be involved in the functional reactivity of the airways as its stimulation can inhibit the contraction of airway smooth muscle. This effect seems to be mediated by the inhibition of acetylcholine release from cholinergic nerves, rather than a direct effect on the smooth muscle itself. Unlike CB₁ receptors, CB₂ receptors are likely to be involved in the mechanisms for neurogenic inflammation, probably acting through the sensory nerves (Figure 1). The contribution of both receptors in the immune modulation of airways is well established, as discussed above. The data of the present literature indicate a significant contribution of CB₂ receptors in allergic diseases, which can be considered for the treatment of allergic asthma. However, the possible involvement of CB₁ receptors should not be excluded, since they are expressed and functional almost in every immune cell. Therefore, appropriate cannabinoid receptor ligands may be rational candidates for the treatment of airway diseases because of their anti-inflammatory and bronchodilatory effects.

Altering the tissue levels of endocannabinoids as a therapeutic strategy is a complex issue. One reason for that is cannabinoids have other targets then CB receptors, which can cause unpredictable effects. The enzymes responsible for endocannabinoid metabolism also have other bioactive by-products, which have significant biological effects. Furthermore, in addition to FAAH or MAGL, other enzymes are also involved in endocannabinoid metabolism, such as COX-2, which leads to the production of various inflammatory prostaglandins. In this respect, the elevation of AEA or 2-AG tissue levels by FAAH or MAGL inhibition were shown to enhance their COX-2-mediated oxidation, and increase prostamide and PG-G signaling [131,132]. These factors make the therapeutic use of FAAH and MAGL inhibitors more complicated, and may partly account for the failure of some FAAH inhibitors like PF-04457845, BIA 10-2474, PF-06818883, V-158866 in clinical trials [6,133,134,135]. For instance, the reasons for the interruption of the clinical trial of BIA 10–2474 due to a death of a volunteer are still not fully established and thought to be related to the off-target effects of FAAH inhibitors [134,135]. However, elevated endocannabinoid levels were not reported to be associated with severe toxic effects on the central nervous system [134]. The specificity of the molecule itself or its metabolite(s) should be considered for the off-target effects, which can cause unpredictable adverse events in clinical trials. Therefore, more studies should be performed in order to clarify the contribution of by-products and other bioactive fatty acids in airway inflammation.