Endocannabinoids in immune regulation and immunopathologies.

Endocannabinoids are key bioactive components of the endocannabinoid system, and the profound influence of endocannabinoids on the modulation of the immune system is being increasingly appreciated. The knowledge of endocannabinoid-immune cell crosstalk will pave the way to therapeutic implications of modulators of this pathway in autoimmune and chronic inflammatory disorders. Endocannabinoids seem to exert both anti-inflammatory and pro-inflammatory effects in specific contexts, based on specific receptor engagement and the downstream signalling pathways involved. In this review, we summarized the biosynthesis, signalling and degradation of two well-studied endocannabinoids-anandamide and 2-arachidonylglycerol in immune cells. Then, we discussed the effects of these two endocannabinoids on the functioning of major innate and adaptive immune cells, along with the choice of receptors employed in such interactions. Finally, we outline our current knowledge on the involvement of anandamide and 2-arachidonylglycerol in context of inflammation, allergies, autoimmunity and metabolic disorders.

Cutting Edge: Dysregulated Endocannabinoid-Rheostat for Plasmacytoid Dendritic Cell Activation in a Systemic Lupus Endophenotype.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease, characterized by loss of tolerance toward self nuclear Ags. Systemic induction of type I IFNs plays a pivotal role in SLE, a major source of type I IFNs being the plasmacytoid dendritic cells (pDCs). Several genes have been linked with susceptibility to SLE in genome-wide association studies. We aimed at exploring the role of one such gene, α/ß-hydrolase domain-containing 6 (ABHD6), in regulation of IFN-α induction in SLE patients. We discovered a regulatory role of ABHD6 in human pDCs through modulating the local abundance of its substrate, the endocannabinoid 2-arachidonyl glycerol (2-AG), and elucidated a hitherto unknown cannabinoid receptor 2 (CB2)-mediated regulatory role of 2-AG on IFN-α induction by pDCs. We also identified an ABHD6High SLE endophenotype wherein reduced local abundance of 2-AG relieves the CB2-mediated steady-state resistive tuning on IFN-α induction by pDCs, thereby contributing to SLE pathogenesis.

Regulation of inflammation by cannabinoids, the endocannabinoids 2-arachidonoyl-glycerol and arachidonoyl-ethanolamide, and their metabolites.

2-Arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA) are endocannabinoids that have been implicated in many physiologic disorders, including obesity, metabolic syndromes, hepatic diseases, pain, neurologic disorders, and inflammation. Their immunomodulatory effects are numerous and are not always mediated by cannabinoid receptors, reflecting the presence of an arachidonic acid (AA) molecule in their structure, the latter being the precursor of numerous bioactive lipids that are pro- or anti-inflammatory. 2-AG and AEA can thus serve as a source of AA but can also be metabolized by most eicosanoid biosynthetic enzymes, yielding additional lipids. In this regard, enhancing endocannabinoid levels by using endocannabinoid hydrolysis inhibitors is likely to augment the levels of these lipids that could regulate inflammatory cell functions. This review summarizes the metabolic pathways involved in the biosynthesis and metabolism of AEA and 2-AG, as well as the biologic effects of the 2-AG and AEA lipidomes in the regulation of inflammation.

DODAB:monoolein liposomes containing Candida albicans cell wall surface proteins: a novel adjuvant and delivery system.

We describe the preparation and characterization of DODAB:MO-based liposomes and demonstrate their adjuvant potential and use in antigen delivery. Liposomes loaded with Candida albicans proteins assembled as stable negatively charged spherical nanoparticles with a mean size of 280 nm. High adsorption efficiency (91.0 ± 9.0%) is attained with high lipid concentrations. The nanoparticles were non-toxic, avidly taken up by macrophage cells and accumulated in membrane rich regions with an internalization time of 20 min. Immunized mice displayed strong humoral and cell-mediated immune responses, producing antibodies (IgGs) against specific cell wall proteins, Cht3p and Xog1p. DODAB:MO-based liposomes loaded with C. albicans proteins have an excellent immunogenic potential and can be explored for the development of an immunoprotective strategy against Candida infections.

Bacterial lipid modification enhances immunoprophylaxis of filarial abundant larval transcript-2 protein in Mastomys model.

As in many other parasitic diseases, efficacious vaccine for lymphatic filariasis has been elusive for want of new approaches leaving billions of people either debilitated or at risk. With multiple B- and T-cell epitopes, the abundant larval transcript-2 (ALT-2) of the filarial worm, Brugia malayi, has been shown to be a promising immunoprophylactic target. To enhance its efficacy, it was lipid modified using our recently developed protein engineering tool, which then offered 30% more immunoprotection (49 vs. 79%) in Mastomys coucha model. Sustained high levels of IFN-γ (about 100 times) and high antibody titres (10-fold) elicited by lipid-modified ALT-2, as compared to the native form, indicated the maintenance of Th1/Th2 balance that is impaired in filariasis. Thus, this study provides the basis for developing efficacious vaccines for filariasis and other parasitic diseases by exploiting bacterial lipid modification.

2-Arachidonoyl-glycerol- and arachidonic acid-stimulated neutrophils release antimicrobial effectors against E. coli, S. aureus, HSV-1, and RSV.

The endocannabinoid 2-AG is highly susceptible to its hydrolysis into AA, which activates neutrophils through de novo LTB(4) biosynthesis, independently of CB activation. In this study, we show that 2-AG and AA stimulate neutrophils to release antimicrobial effectors. Supernatants of neutrophils activated with nanomolar concentrations of 2-AG and AA indeed inhibited the infectivity of HSV-1 and RSV. Additionally, the supernatants of 2-AG- and AA-stimulated neutrophils strongly impaired the growth of Escherichia coli and Staphylococcus aureus. This correlated with the release of a large amount (micrograms) of α-defensins, as well as a limited amount (nanograms) of LL-37. All the effects of AA and 2-AG mentioned above were prevented by inhibiting LTB(4) biosynthesis or by blocking BLT(1). Importantly, neither CB(2) receptor agonists nor antagonists could mimic nor prevent the effects of 2-AG, respectively. In fact, qPCR data show that contaminating eosinophils express ∼100-fold more CB(2) receptor mRNA than purified neutrophils, suggesting that CB(2) receptor expression by human neutrophils is limited and that contaminating eosinophils are likely responsible for the previously documented CB(2) expression by freshly isolated human neutrophils. The rapid conversion of 2-AG to AA and their subsequent metabolism into LTB(4) promote 2-AG and AA as multifunctional activators of neutrophils, mainly exerting their effects by activating the BLT(1). Considering that nanomolar concentrations of AA or 2-AG were sufficient to impair viral infectivity, this suggests potential physiological roles for 2-AG and AA as regulators of host defense in vivo.

Involvement of the endogenous cannabinoid 2 ligand 2-arachidonyl glycerol in allergic inflammation.

BACKGROUND: Cannabinoid (CB) 2 is expressed on immune and inflammatory cells. Identification of 2-arachidonyl glycerol (2-AG) and anandamide as endogenous CB2 ligands has allowed investigations of the roles of CB2 and its endogenous ligand system in inflammatory cells. However, the roles of this receptor-ligand system in inflammatory and allergic immune responses in vivo have not been fully elucidated. METHODS: Two mouse allergy models, namely ear dermatitis induced by 2,4-dinitrofluorobenzene and allergic bronchitis induced by ovalbumin, were analyzed for 2-AG amounts in allergic tissues, with reference to allergic and inflammatory symptoms. To investigate the gene expression via CB2 in inflammatory cells, human promyelocytic HL-60 cells were stimulated by the CB2 ligand 2-AG ether and analyzed using a DNA microarray. RESULTS: In the ear dermatitis model, the 2-AG amount increased upon serial 2,4-dinitrofluorobenzene challenges and was correlated with ear weight gain. The increased ear thickness in this allergy model was clearly suppressed in CB2 knockout mice, suggesting that the generated endogenous CB2 ligands induce ear thickness through aberrant inflammatory responses and remodeling mediated via CB2. In the allergic bronchitis model, the 2-AG level in bronchoalveolar lavage was increased and sustained during the elevation of inflammatory cell infiltration. The DNA microarray analysis of human HL-60 cells revealed that 2-AG ether induced expressions of not only inflammatory chemokines/cytokines but also of cell growth factors. CONCLUSION: Our data strongly suggest that endogenous CB2 ligands upregulated upon disease progression in allergic models are involved in aberrant alterations of both inflammatory responses and tissue cell growth.

Involvement of cannabinoid receptor CB2 in dectin-1-mediated macrophage phagocytosis.

Cannabinoid receptors are expressed in macrophages, but little is known of their roles. We here examined their involvement in phagocytosis. The presence of 2-arachidonylglycerol, an endocannabinoid, augmented the phagocytosis of zymosan by mouse macrophages, while the phagocytosis of Escherichia coli, Staphylococcus aureus, apoptotic cells or latex beads remained unaffected. An agonist of the cannabinoid receptors CB1 and CB2 also stimulated the phagocytosis of zymosan. The stimulatory effect of 2-arachidonylglycerol was abolished when phagocytosis reactions were carried out in the presence of an antagonist of CB2 but not of CB1. Furthermore, the phagocytosis of zymosan in the presence of 2-arachidonylglycerol was severely inhibited by the addition of a beta-glucan-containing carbohydrate or antibody neutralizing dectin-1, a beta-glucan-recognizing phagocytosis receptor. These results suggested that the activation of CB2 in macrophages leads to the stimulation of dectin-1-mediated phagocytosis.

Reduced endocannabinoid immune modulation by a common cannabinoid 2 (CB2) receptor gene polymorphism: possible risk for autoimmune disorders.

Immune system responsiveness results from numerous factors, including endogenous cannabinoid signaling in immunocytes termed the "immunocannabinoid" system. This system can be an important signaling pathway for immune modulation. To assess the immunomodulating role of the cannabinoid 2 (CB2) receptor, we sought polymorphisms in the human gene, identified a common dinucleotide polymorphism, and investigated its effect on endocannabinoid-induced inhibition of T lymphocyte proliferation. The CB2 cDNA 188-189 GG/GG polymorphism predicts the substitution of glutamine at amino acid position 63 by arginine. T lymphocytes from CB2 188-189 GG/GG homozygotes had approximately twofold reduction of endocannabinoid-induced inhibition of proliferation compared with cells from CB2 188-189 AA/AA homozygotes. In GG/GG subjects, the reduced endocannabinoid inhibitory response was highly significant for N-arachidonylglycine and nearly significant for 2-arachidonylglycerol, and a specific CB2 receptor antagonist partially blocked these effects. Also, patients with autoimmune diseases had an increased prevalence of the homozygous GG/GG genotype. Collectively, these results demonstrate reduced endogenous fatty acid amide immunomodulatory responses in individuals with the CB2 188-189 GG/GG genotype and suggest that this CB2 gene variation may be a risk factor for autoimmunity. The results also support the proposition that the CB2 receptor may represent a novel pharmacological target for selective agonists designed to suppress autoreactive immune responses while avoiding CB1 receptor-mediated cannabinoid adverse effects.

Oral immunisation of chickens using cholera toxin B subunit and Softigen as adjuvants results in high antibody titre in the egg yolk.

Oral immunisation by gavage of laying hens with human immunoglobulin G (IgG) combined with a number of potential adjuvants was performed. The resulting immunospecific egg yolk (IgY) antibodies were quantified by ELISA. The following adjuvants were tested: A Poly(lactide-co-glycolide) (PLG) microspheres, Cholera toxin B-subunit (CTB), CTB conjugated with glutaraldehyde, Dimethyl dioctadecyl ammonium bromide (DDA), and Softigen (pegylated C8/C10 mono/di glyceride). Hens in a positive control group were immunised with human IgG in saline emulsified with an equal volume of Freund's Incomplete Adjuvant. High titres of immunospecific IgY antibodies against human IgG were recorded in the eggs from the chickens immunised orally with the antigen combined with glutaraldehyde conjugated CTB and in the chickens immunised with the antigen combined with Softigen. The present results show that invasive technique related stress could be eliminated/reduced in polyclonal antibody producing animals.

Nasal and parenteral immunizations with diphtheria toxoid using monoglyceride/fatty acid lipid suspensions as adjuvants.

A novel suspension system was developed where monoglycerides were formulated together with fatty acids and subsequently admixed with antigens. In the present study, diphtheria toxoid was used as a model antigen primarily due to its weak immunological properties as well as to its importance as a future human vaccine for mucosal, particularly nasal immunization. The formulations were administered parenterally and/or nasally to mice whereafter the immune response was determined. In the present study, we have shown that mono-olein/oleic acid vesicles enhance the immunogenicity of admixed diphtheria toxoid in mice to the same level as Alum adsorbed (or Freund's complete adjuvant) when administered parenterally or nasally. It was also shown that the immunogenicity was linked to the length of the acyl chain of the lipids, where shorter acyl chains resulted in reduced titers. Furthermore, shorter acyl chains also gave rise to more pronounced toxic reactions at the injections sites, such as necrosis and alopeci, both of which were lacking when the optimal formulation consisting of mono-olein and oleic acid was used. Thus, this lipid matrix has in our view a great potential as an immunological adjuvant with an exceptionally simple and efficient preparation procedure without organic solvents and with low cost endogenous lipid based raw materials.

Plant immunomodulators for termination of unwanted pregnancy and for contraception and reproductive health.

Neem (Azadirachta indica) seed and leaf extracts have spermicidal, anti-microbial, anti-fungal and anti-viral properties. They are also immunomodulators that induce primarily a TH1 type response. These properties are being exploited to develop two different useful methods of fertility control. Neem extracts given orally at early post-implantation stage terminate pregnancy in rodents and primates. Treatment has no residual permanent effect and fertility is regained in subsequent cycles. The mechanism by which the action occurs is not fully clear. A transient increase in CD4 and more significantly in CD8 cells is noticed in mesenteric lymph nodes and spleen. A rise in immunoreactive and bioactive TNF-alpha and IFN-gamma in draining lymph nodes, serum and foetal-placental tissue is observed. A polyherbal cream and pessary have been developed containing three active ingredients of plant origin. These have synergistic spermicidal properties on human sperm as determined by the Sander Cramer test. Their use before mating has high contraceptive efficacy in rabbits and baboons. Another interesting property is their inhibitory action on a wide spectrum of micro-organisms, including Candida albicans, C. tropicalis, Neisseria gonorrhoeae, the multidrug-resistant Staphylococcus aureus and urinary tract Escherichia coli, Herpes simplex-2 and HIV-1. Phase I clinical trials have been completed in India, Egypt and the Dominican Republic, and indicate the safety of the formulation, its acceptability and beneficial action invaginosis due to infections.

The adjuvant activity of non-ionic surfactant vesicles (niosomes) on the BALB/c humoral response to bovine serum albumin.

The ability of non-ionic surfactant vesicles (NISV) to enhance antibody production against bovine serum albumin (BSA) was compared with Freund's complete adjuvant (FCA), in the BALB/c mouse. Two subcutaneous inoculations with NISV entrapped BSA induced antibody levels comparable to, and persisting as long as those produced by FCA by either the subcutaneous or intraperitoneal route of inoculation. Intraperitoneal inoculation of NISV did not generate as strong an antibody response. The adjuvant activity of NISV was wholly dependent on the BSA being entrapped within preformed vesicles; mixing free BSA with vesicles was not effective. Analysis of the anti-BSA IgG subclasses induced by NISV and FCA showed that NISV were generally better stimulators of IgG2a than was FCA, but poorer stimulators of IgG1. From this, we deduce that NISV are potentially better stimulators of the Th1 lymphocyte subset than is FCA and by inference, potent stimulators of cellular immunity. We believe that NISV may offer many advantages over other adjuvants in terms of immunological selectivity, low toxicity and stability.

[Glycerol monothioglycolate eczema in a hairdresser. Persistence of the allergen in the hair several weeks after the application of a permanent]. / Eczéma au monothioglycolate de glycérol chez une coiffeuse. Persistance de l'allergène dans les cheveux plusieurs semaines après l'application d'une permanente.

Glyceryl thioglycolate has been used for a pair of years in acid permanent wave solutions. It appears to be much more sensitizing than ammonium thioglycolate which is usually the main component of this kind of cosmetics. A case of allergic contact dermatitis due to glyceryl thioglycolate is reported. The patient was a 43-year old woman with an eczematous dermatitis of both hands. She was a hairdresser and eczematous eruptions were strictly work-related. She had no direct contact with most cosmetics and only used tools (scissors, clips, pins, rollers, combs...) and hair spray. Patch testing was however done with the standard patch-test screening tray and all the products used in the shop. The only positive reaction was due to a permanent wave solution containing only glyceryl thioglycolate and glycerol. Positive patch-test reactions were also obtained with hair treated with the permanent wave solution and cut immediately or several weeks after the application of the preparation. This observation shows that glyceryl thioglycolate may be responsible for allergic contact dermatitis in hairdressers; it emphasizes the long remanence of the allergen in the hair that makes its eviction specially difficult.

Induction of EL4 cell resistance to syngeneic macrophage-mediated lysis by protein kinase C ligands; effects of cultured TPA-treated target cell and protein phosphorylation.

Pretreatment of EL4 cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) for 30 min renders them resistant to lysis by activated macrophages (M phi). This resistance was augmented two to three-fold when TPA-treated EL4 cells were incubated for 2-6 hr prior to co-culture with M phi. Preincubation of TPA-treated cells for 24 hr could result in 100% resistance. in this paper we show that an endogenous ligand for protein kinase C, oleoyl-2-acetate glycerol (OAG), was capable of inducing tumour cell resistance to M phi kill and, similar to the effects seen with TPA, OAG did not affect the selective binding of tumour cells to activated M phi. Another important observation on the mechanism of TPA induction of tumour cell resistance was that once the target cells were programmed to die after a minimal contact with activated M phi of 4-6 hr, TPA treatment was ineffective in altering the percent lysis 20 hr later. To investigate whether any possible correlation exists between TPA-induced protein phosphorylation and acquisition of resistance, EL4 cells were labelled with 32P and treated simultaneously with TPA, and cellular proteins were resolved by two-dimensional gel electrophoresis. Eight polypeptides (MW 24,000-70,000, pI 4.8-6.1) showed consistent increased phosphorylation as a result of TPA treatment. One-minute exposure with TPA resulted in enhanced phosphorylation of only four peptides (MW 39,000, 58,000, 63,000, 70,000) while all eight polypeptides showed increased phosphorylation by 10 min.

[Somatomedin activity of the blood: hormonal metabolic and immunological shifts in cancer patients]. / Somatomedinovaia aktivnost' krovi: gormonal'no-metabolicheskie i immunologicheskie sdvigi u onkologicheskikh bol'nykh.

Blood somatomedin activity observed in patients with cancer of the corpus uteri, breast, colon and rectum, pancreas, ovary and lung proved to exceed those in control by 140-300%. The said parameter was found to depend upon endocrine-metabolic changes, relatively higher levels being registered in overweight patients and those with hypertriglyceridemia. Changes of varying degree in somatomedin activity and growth hormone levels produced by insulin and glucose in patients with breast and uterine cancer pointed to hormonal dependence of the parameter. Also, the effect is a circumstantial evidence for negative feedback between growth hormone and somatomedin production. In patients with cancer of the breast, uterus and lung, a significant correlation was established between somatomedin activity, on the one hand, and monocyte. B-lymphocyte and T-active lymphocyte levels, on the other, matched by an inverse one with T-lymphocyte level and blastogenic reaction of lymphocytes.