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INFLAMMATION AND IMMUNOPHARMACOLOGY
Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy
Received March 19, 2004; accepted June 1, 2004.
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Abstract |
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) and experimental animals (Coffey et al., 1996). More recently, the effects of THC on human immune functions and host defense have been reviewed, providing evidence that THC can suppress the human immune response in a host of leukocyte subsets and alveolar macrophages, further stressing the relationship between marijuana smoking and immunologically-related diseases (Klein et al., 1998).
Of the immunocompetent cells, mast cells are strategically placed in tissues that interface with the external environment and vary in the way in which their activation is modified by endocannabinoids. In the rat ear pin, the degranulation of resident mast cells induced by substance P is fully abrogated by the endogenous ligands at cannabinoid (CB) receptors arachidonylethanolamide (anandamide; AEA) and palmitoylethanolamide (Aloe et al., 1993). Our preliminary results showed that 2-arachidonylglycerol (2AG), a CB2 receptor ligand (Sugiura et al., 2000), significantly reduced the immunological release of histamine from guinea pig mast cells (Vannacci et al., 2002). Furthermore, endocannabinoids down-regulate the immunological activation of the RBL-2H3 mast cell line, acting through CB2 receptor-mediated AKT and extracellular signal-regulated kinase phosphorylation (Samson et al., 2003; Vannacci et al., 2003a). The fact that mast cells themselves produce endocannabinoids, including 2AG, suggests that an autocrine loop exists (Bisogno et al., 1997). However, recent evidence shows that endocannabinoids do not influence the releasability of rat peritoneal mast cells (Lau and Chow, 2003), casting some doubt on the overall effects of cannabinoids.
The prevalent down-regulation of mast cell releasability reported in the literature could be solely accounted for by the activation of CB2 receptors or conceivably imply that there is autocrine generation of inhibitory mediators. Among them, nitric oxide (NO) and prostanoids seem to be linked to the cannabinoid system. The link between endocannabinoids and the NO pathway has been frequently reported. AEA stimulates NO release from human monocytes that was inhibited by N-monomethyl-L-arginine methylester (L-NAME), a non-selective inhibitor of NO synthase (NOS) (Stefano et al., 1996). A stereospecific binding site for AEA is present in Mytilus edulis immunocytes, coupled with NO release from these cells (Stefano et al., 1997). In addition to the immunocompetent cells, AEA is coupled with the NO pathway in invertebrate microglia (Stefano et al., 1996), human saphenous vein endothelium (Stefano et al., 1998), rat brain median eminence (Prevot et al., 1998), and human arterial endothelial cells (Fimiani et al., 1999b), where it stimulates NO release. It seems likely, therefore, that the CB1 receptor ligand AEA could act, at least in part, via the generation of NO. However, in regard to peripheral CB2 receptors, no studies have addressed the interaction between cannabinoids and the NO pathway on the response of guinea pig mast cells to allergic stimuli using endogenous and/or exogenous CB2 receptor agonists and antagonists.
Modulation of prostaglandin (PG) formation by cannabinoids has been widely established. The metabolically stable analog of AEA methanandamide induces cyclooxygenase (COX) expression in human microglioma (Ramer et al., 2001) and murine lung cancer cells (Gardner et al., 2003). In addition to the induction of the generating enzyme, AEA has been shown to release arachidonic acid and generate PGF2
in neuronal cells in culture (Someya et al., 2002). Other interactions between the prostanoid and cannabinoid systems entail the indomethacin (INDO)-induced shift of arachidonic acid metabolism toward endocannabinoid synthesis, secondary to COX inhibition (Guhring et al., 2002) and the susceptibility of endocannabinoids to oxidative metabolism via COX (Kozak et al., 2000). However, no studies have explored the interaction between CB2 receptors and the prostanoid pathway using an endogenous agonist and selective agonists and antagonists at CB2 receptors.
The first aim of the present study was to evaluate the effects of endogenous and exogenous ligands at CB2 receptors on the immunological activation of guinea pig mast cells. The second aim was to address whether the effect of CB2 receptor activation could be modulated by manipulation of NO and prostanoid pathways.
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Materials and Methods |
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Isolation of Serosal Mast Cells from Actively Sensitized Guinea Pigs. Male albino Dunkin-Hartley guinea pigs (300–350 g body weight) were used. They were purchased from a commercial dealer (Rodentia, Bergamo, Italy) and quarantined for 7 days at 22 to 24°C with a 12-h light/dark cycle before use. The experimental protocol was designed in compliance with the guidelines of the European Community (86/609/EEC) for animal care and the use of laboratory animals and was approved by the Animal Care Committee of the University of Florence in agreement with Good Laboratory Practice.
Guinea pigs were sensitized with ovalbumin (100 mg kg-1 i.p. plus 100 mg kg-1 s.c.) suspended in saline (40 mg ml-1). The animals were anesthetized with ethyl ether and decapitated 18 to 21 days after sensitization. Mast cells were obtained as previously described (Vannacci et al., 2002). The peritoneal and pleural cavities were washed for 30 min with 15 and 5 ml, respectively, using a solution of the following composition: 137 mM NaCl, 5.6 mM glucose, 2.7 mM KCl, 0.3 mM NaH2PO4, 1 mM CaCl2, 10 mM HEPES, and 1 mg ml-1 collagenase. Mast cells were then isolated by density gradient centrifugation on Ficoll and washed twice with a medium of the following composition: 145 mM NaCl, 2.4 mM KCl, 0.9 mM CaCl2, and 0.1% glucose adjusted to pH 7.4 with 10% Sörensen phosphate buffer. The procedure yielded a cell population composed of 95% mast cells. The cells were suspended in 2 ml of the same medium as reported before and then exposed to the drugs under study for 30 min at 37°C and stimulated for 30 min with antigen (ovalbumin, 100 µg ml-1). When an antagonist was used, cells were preincubated for 30 min with the antagonist and then for a further 30 min with the agonist. The reaction was stopped by chilling the tubes in an ice-water bath. The cells were then centrifuged at 500g (15 min, 4°C), and histamine was measured in the supernatants and in the pellets.
Histamine Release Assay. Histamine was measured fluorometrically in mast cell suspensions treated as described above and stimulated or not with 100 µg ml-1 ovalbumin, using the previously described method (Ndisang et al., 1999). Briefly, the samples were centrifuged at 200g (10 min, 20°C). In the supernatants, 0.1% O-phthaldialdehyde (in methanol) was added directly to the samples after alkalinization with 0.5 ml of 0.5 N NaOH. The reaction was stopped after 4 min by adding 0.2 ml of 2.5 N H3PO4. The same procedure was used for the pelleted cells after extraction with 0.1 M HCl. Histamine in the samples was determined by fluorometric measurement using an excitation wavelength of 365 nm and an emission wavelength of 455 nm. Histamine release (supernatant histamine) was expressed as a percentage of the total present in the cells plus supernatants.
Evaluation of NO Production. Evaluation of NO production was performed by determining the nitrite (
) amount, the stable end products of NO metabolism, in guinea pig mast cell supernatants obtained as described. In some experiments, the cells were pretreated for 30 min with 10 µM L-NAME, an NOS inhibitor, before incubation with the tested substances (2AG and CP55,940) or with medium alone. The amount of in cell supernatants was measured spectrophotometrically by the Griess reaction. Briefly, samples were supplemented with 276 mU of nitrate reductase and 40 µM NADPH+ and then allowed to react with the Griess reagent (aqueous solution of 1% sulfanylamide and 0.1% naphthylethylendiamine di-hydrochloride in 2.5% H3PO4) to form a stable chromophore absorbing at a wavelength of 546 nm. The values were obtained by comparison with reference concentrations of sodium nitrite and expressed as net amounts of nitrite (nanomoles) per milligram of protein (Salvemini et al., 1991). The protein concentrations (milligrams per milliliter) were determined by the Lowry method using BSA as standard (Lowry et al., 1951).
Evaluation of PGE2 Production. Prostaglandin E2 levels were determined by a competitive enzyme immunoassay kit in guinea pig mast cell supernatants obtained as described and added with 10 µM indomethacin to inhibit the formation of COX products. In some experiments, the cells were pretreated for 30 min with 100 nM SR144528, a CB2 blocker, 10 µM indomethacin, an unselective COX blocker, and 100 nM rofecoxib, a selective COX-2 blocker, before 30 min incubation with the tested substances (2AG and CP55,940) or with medium alone. Each sample was assayed in triplicate, and the values were normalized with respect to protein concentrations. The protein concentrations (milligrams per milliliter) were determined as previously reported. The values were expressed as picograms of PGE2 per milligram of protein.
Determination of Intracellular Calcium Concentration ([Ca2+]i). Isolated mast cells were suspended in a buffer containing 20 mM HEPES, 127 mM NaCl, 50 mM KCl, 0.1 mg ml-1 glucose, and 1% BSA, adjusted to pH 7.4, and loaded with 3 mM Fura-2/AM for 1 h in a shaking water bath at 37°C. At the end of the incubation, mast cells were centrifuged at 500g (15 min, 4°C), and the supernatant was discarded. Cells were then washed twice with the buffer previously reported, aliquoted into the samples, and incubated at 37°C in a shaking water bath for 30 min with the drugs under study. When an antagonist was used, cells were preincubated for 30 min with the antagonist and then for a further 30 min with the agonist. Cytosolic-free Ca2+ levels were determined spectrofluorometrically using a Shimadzu DR 15 spectrofluorometer (Shimadzu, Kyoto, Japan), which allows the measurement of both peak and plateau values. The fluorescence excitation spectrum was scanned at wavelengths ranging from 300 to 420 nm, with an emission wavelength fixed at 510 nm. The values of cytosolic Ca2+ levels ([Ca2+]i) were calculated by a computer program using the following equation: [Ca2+]i = Kd[(F - Fmin)/(Fmax - F)], where Fmin and Fmax are the fluorescence values at very low and high Ca2+ concentrations, respectively. Fmin was obtained by measuring fluorescence in the presence of 8 mM EGTA, and Fmax was obtained by measuring fluorescence in digitonin-lysed samples. A Kd value of 224 nM was used for the apparent dissociation constant of Fura-2/AM (Ndisang et al., 1999).
Radioimmunoassay of Cyclic Nucleotides. The concentrations of cGMP were determined by means of radioimmunoassay using 125I-labeled cyclic nucleotides as previously described (Ndisang et al., 1999). A suspension of 105 guinea pig mast cells in the presence of 10-4 3'-isobutyl-1-methylxanthine was diluted with Krebs buffer containing 137 mM NaCl, 2.7 mM KCl, 11.9 mM NaHCO3, 0.3 mM Na2HPO4, 0.8 mM MgSO4, 5.6 mM glucose, and 1 mM CaCl2. Indomethacin (10 µM) was added to the samples to inhibit the formation of COX products. At the end of the experiment, 500 µl of 10% w/v trichloroacetic acid were added to each sample containing mast cells, and the cyclic nucleotides were extracted from trichloroacetic acid with 0.5 M tri-n-octylamine dissolved in 1,1,2-trichloro-trifluoroethane. Finally, the samples were acetylated with acetic anhydride, and the amounts of cGMP in the aqueous phase were measured by radioimmunoassay. The values were expressed as fem-tomoles of cGMP per milligram of protein. The protein concentrations were determined as previously described.
Western Blotting Inducible NOS (iNOS) and COX. Guinea pig mast cells obtained as described were incubated for 60 min with CP55,940 to evaluate the expression of iNOS and COX (COX-1 and -2). The cells were lysed in ice-cold buffer [0.9% NaCl, 20 mM TRIZMA-HCl (pH 7.6), 0.1% Triton X-100, 1 mM PMSF, and 0.01% leupeptin] and centrifuged at 10,000g (10 min, 4°C). The protein content of the supernatants was determined as previously described. The cell lysate was mixed 1:1 with sample buffer [20 mM TRIZMA-HCl (pH 6.8), 20% glycerol, 2% SDS, 5% mercaptoethanol, and 0.025% bromophenol blue] and boiled (Pang and Hoult, 1997). SDS-polyacrylamide gel electrophoresis was performed using 8 and 5% acrylamide for the separating and stacking gel, respectively. Proteins were transferred to nitrocellulose membrane. Blots were blocked with 5% Blocker BSA solution in phosphate-buffered saline (Pierce) and incubated overnight at 4°C with the primary antibodies. Blots were further incubated with secondary antibodies conjugated with horseradish peroxidase for 2 h at room temperature and, finally, incubated with SuperSignal West Pico Chemiluminescent Substrate (Pierce) for 5 min and exposed to CL-Xposure Film. The HCT116 cell line served as a positive control for iNOS (Jenkins et al., 1994), and the HT29 cell line served as a positive control for COX-1 and COX-2 (Liu et al., 2003). Quantitative analysis was performed by means of Scion Image Beta 4.02 freeware software (Scion Corporation, Frederick, MD; http://www.scioncorp.com); all values were normalized to actin and to the respective basal levels.
Statistical Methods. Statistical analysis was performed using SPSS statistical software (Release 11.0; SPSS Inc., Chicago, IL). Groups were compared using Student's t test for unpaired values or the Kruskal-Wallis H test followed by the Mann-Whitney U test, when appropriate; p values equal to or less than 0.05 were considered statistically significant.
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) on rat peritoneal mast cells. However, when the incubation was carried out in the presence of PMSF (100 µM, 30-min preincubation), AEA and 2AG were effective in decreasing the immunological release of histamine, showing that preserving the hydrolysis of cannabinoids by blocking the fatty acid amide hydrolase with PMSF was crucial to studying their effects. In fact, in the presence of PMSF, 2AG decreased the immunological release of histamine, starting in a concentration-dependent manner at 1 nM (Fig. 1, panel a). The effect was dose-dependently reduced by the CB2 blocker SR144528 (10 and 100 nM, 30-min preincubation) and left unchanged by AM251 (100 nM, 30-min preincubation), a selective antagonist at CB1 receptors, suggesting that the down-regulation of antigen-induced histamine release was mediated by CB2 receptors (Fig. 1, panel a). The participation of CB2 receptors was strengthened by the observation that the compound CP55,940 (10 nM–1 µM), an agonist at CB receptors (Iversen and Chapman, 2002), mimicked the inhibitory action of the endogenous ligand, and this effect was abrogated by SR144528, a selective CB2 receptor antagonist, and not by AM251 (Fig. 1, panel b). Neither PMSF, SR144528, nor DMSO modified the basal and antigen-induced histamine levels (data not shown).
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When the incubation with 2AG (1–100 nM) was carried out in the presence of L-NAME (10 µM, 30-min preincubation), an inhibitor of NO synthase, the decrease in the release of histamine was significantly reduced, and it was reinstated in the presence of the physiological substrate L-arginine (10 µM), suggesting that NO participates in the down-regulation of the immunological activation of mast cells (Fig. 2, panel a). Accordingly, L-NAME also reduced the inhibitory effect of the CB2 receptor agonist CP55,940 (10 nM–1 µM), and L-arginine reinstated the inhibitory effect (Fig. 2, panel b). Neither L-NAME nor L-arginine was capable of modifying the basal and antigen-induced histamine release. Consistent with these effects is the observation that 2AG (10 nM–1 µM) determined a concentration-dependent generation of NO from mast cells, as shown by the evaluation of the nitrite production, which was abated by preincubating the cells with L-NAME (10 µM, 30-min preincubation; Fig. 3, panel a). The iNOS selective inhibitor 1400W (100 nM, 30-min preincubation) also significantly reduced nitrite generation from mast cells, suggesting an involvement of the inducible isoform of NOS (Fig. 3, panel a). Interestingly, CP55,940 (1 nM–1 µM) also increased the nitrite production from mast cells in a way that was abrogated by L-NAME (10 µM, 30-min preincubation) or 1400W (100 nM, 30-min preincubation) (Fig. 3, panel b). Furthermore, CP55,940 (1 µM, 60-min incubation) induced the expression of iNOS protein, as shown by Western blot analysis (Fig. 3, panels c and d). PMSF, L-NAME, or 1400W alone were not capable of modifying the nitrite levels (data not shown).
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Manipulation of the synthesis of prostanoids also modulated the effect of endogenous and exogenous cannabinoids on antigen-induced histamine release from mast cells. Indeed, either blocking COX-1 and -2 with indomethacin (10 µM, 30-min preincubation) or blocking COX-2 with rofecoxib (ROFE; 100 nM, 30-min preincubation) significantly reduced the inhibition of the release of histamine induced by both 2AG and CP55,940 (Fig. 4, panels a and b). Indomethacin and rofecoxib did not change the basal and antigen-induced release of histamine (data not shown). Incubation of mast cells with 2AG or CP55,940 (1 nM–1 µM) elicited an increased production of PGE2; this effect was completely inhibited by the CB2-selective antagonist SR144528 (100 nM, 30-min preincubation), indomethacin (10 µM, 30-min preincubation), and rofecoxib (100 nM, 30-min preincubation) (Fig. 5, panels a and b). A Western blot analysis showed that COX-1 protein was constitutively expressed in guinea pig mast cells, whereas COX-2 protein was expressed only after the treatment with CP55,940 (1 µM, 60-min incubation) (Fig. 5, panel c).
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The release of histamine from mast cells is a calcium-dependent process (Foreman et al., 1977). Accordingly, mast cell stimulation with antigen showed a striking increase in the [Ca2+]i levels (Fig. 6). When mast cells were exposed to 2AG (10 nM) before antigen challenge, a significant decrease in [Ca2+]i was observed, which was abrogated preferentially by SR144528 (100 nM, 30-min preincubation) and less consistently by L-NAME (10 µM, 30-min preincubation; Fig. 6, panel a). The same results were obtained with the exogenous cannabinoid ligand CP55,940 (1 µM) (Fig. 6b). Neither SR144528 nor L-NAME alone was capable of modifying [Ca2+]i levels (data not shown).
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Our previous results have shown that when mast cells from actively sensitized guinea pigs were challenged with the antigen, there was a significant increase in cAMP levels, whereas cGMP levels were unaffected (Ndisang et al., 1999). In unstimulated mast cells, the exposure to 2AG or to CP55,940 significantly raised cGMP concentrations. The increased levels of cGMP were abated by blocking CB2 receptors with SR144528 (100 nM, 30-min preincubation) or inhibiting NOS and COX pathways with L-NAME (10 µM, 30-min preincubation), indomethacin (10 µM, 30-min preincubation), and rofecoxib (100 nM, 30-min preincubation; Fig. 7). SR144528, L-NAME, indomethacin, and rofecoxib alone did not modify the levels of cGMP (data not shown).
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Discussion |
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These data suggest the presence of functional CB2 receptors on mast cells; however, the function of CB2 receptors on mast cells is uncertain. Following the demonstration that palmitoyl-ethanolamide abrogates the peptidergic degranulation of rat mast cells in vivo (Aloe et al., 1993), the same authors demonstrated that rat peritoneal mast cells express both the gene and the protein of a CB2 receptor (Facci et al., 1995). These data were criticized on the basis that the secretion of histamine induced by THC in rat mast cells was not antagonized by SR144528 (Bueb et al., 2001). In line with this observation, Lau and Chow (2003) have recently shown that cannabinoid receptor agonists do not modify the immunological histamine release from rat mast cells, although their experiments were carried out in the absence of the inhibition of fatty acid amide hydrolase.
On the other hand, our experiments are in keeping with previous reports on the anti-inflammatory effects of N-acethylethanolamines (Ganley et al., 1958) and the involvement of endocannabinoids in controlling mast cell activation (Aloe et al., 1993). They also suggest that the down-regulation of the immunological activation of guinea pig mast cells afforded by both endogenous (2AG) and exogenous (CP55,940) cannabinoids entails selective CB2 receptor activation.
However, the concentration-dependent inhibitory effect of both compounds could also be attributable to CB1 receptors, since 2AG and CP55,940 are known to behave as unselective agonists at both receptor subtypes (Barth and Rinaldi-Carmona, 1999). This is not the case, since compound SR144528, a selective CB2 receptor antagonist, concentration-dependently abated their effects, whereas the compound AM251, a CB1 receptor antagonist, was ineffective. These findings strengthen the prevailing involvement of CB2 receptors.
The present experiments also show that the stimulation by 2AG and CP55,940 of the G-protein-coupled CB2 receptors could parallel a variety of intracellular events that are relevant in understanding the inhibitory effect. In fact, the intracellular signals set in motion by 2AG and CP55,940 entail the production of NO and PGE2 involving the induction of iNOS and COX-2, the increase in the intracellular levels of cGMP, and the blunting of the antigen-induced increase in intracellular calcium. The link between endocannabinoids and the NO pathway is well established by experiments showing that AEA stimulates the generation of NO in a host of experimental conditions, both in vivo and in vitro (Stefano et al., 1996, 1997, 1998; Prevot et al., 1998; Fimiani et al., 1999a). The present experiments add further evidence to the relationship between the endocannabinoids and the NO system by showing that 2AG and CP55,940 generate NO from guinea pig mast cells. Gilchrist et al. (2002) have recently described in rat mast cells the presence of a functioning endothelial NOS system and the ability to produce NO through iNOS upon exposure to various immunological stimuli. In our experiments, the use of the iNOS-specific inhibitor 1400W and the Western blot analysis present evidence for a cannabinoid-induced expression of iNOS. Nitrite production seems to be more evident upon treatment with CP55,940 than with 2AG; nevertheless, further studies are needed to ascertain whether this effect is due to the lability of the endogenous cannabinoid or to different pharmacodynamic properties.
Nitric oxide generated via iNOS induction (Salvemini et al., 1991; Vannacci et al., 2003b) or released by NO donors (Masini et al., 1994) has been shown to inhibit the immunological and nonimmunological response of guinea pig and rat mast cells, even if the mechanism by which NO modulates mast cell activation is still a matter of debate (Brooks et al., 1999). Our previous studies demonstrated an involvement of the guanylate cyclase/cGMP system and the decrease of the intracellular calcium available for the exocytosis (Ndisang et al., 1999). Also, in the present experiments, 2AG and CP55,940 increased the intracellular levels of cGMP and blunted the rise of the intracellular calcium induced by antigen challenge. The effects were abrogated by SR144528, L-NAME, and COX-1 and COX-2 inhibitors, suggesting that the generation of NO and PGE2 could in turn inhibit the immunological activation of mast cells by raising the intracellular cGMP and decreasing the amount of calcium necessary for the exocytosis (Ndisang et al., 1999).
The links between the prostanoid and cannabinoid systems are less consistent. In any case, the present experiments clearly show that 2AG and CP55,9440 increase the generation of PGE2 from mast cells, an effect linked to COX-1 and the induction of COX-2 expression.
Whether the increased generation of PGE2 has an inhibitory effect on mast cell activation is a debated issue. In rat mast cells, PGE1 inhibits the immunological release of histamine (Kaliner, 1979). In the same cells, using a different secretagogue (compound 48/80), PGE1 inhibits the release of histamine, although at rather high concentrations (Loeffler et al., 1971). Ennis et al. (1983) have shown that low concentrations of PGD2 and PGE1 are without significant effects on the immunological release of histamine from rat peritoneal mast cells, in contrast with the reported inhibition of the anaphylactic histamine release with low doses of PGD2. In addition, it has been shown that high concentrations of PGD2 inhibit histamine release only in combination with phosphodiesterase inhibition (Wescott and Kaliner, 1981).
In conclusion, circumstantial evidence suggests that the mechanism by which CB2 ligands modulate mast cell activation is by generating NO and PGE2, acting in an autocrine manner in the inhibition of allergic histamine release. As an alternative explanation, the activation of CB2 receptors and the generation of NO and PGE2 could be separate events. However, also in this case, 2AG may be considered an endogenous inhibitor of mast cell activation, and the CB2 receptor agonists may be considered as potential therapeutic drugs useful for controlling inflammation and modulating tissue immune response.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: THC, tetrahydrocannabinol; CB, cannabinoid; AEA, anandamide; 2AG, 2-arachidonylglycerol; NO, nitric oxide; L-NAME, NG-monomethyl-L-arginine methylester; NOS, nitric-oxide synthase; PG, prostaglandin; COX, cyclooxygenase; INDO, indomethacin; OA, ovalbumin; PMSF, phenylmethylsulfonylfluoride; BSA, bovine serum albumin; CP55,940, (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol; AM251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; 1400W, N-(3-(aminomethyl)benzyl)acetamidine; SR144528, N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide; DMSO, dimethyl sulfoxide; iNOS, inducible nitric-oxide synthase; ROFE, rofecoxib.
1 Current address: Department of Internal Medicine, University of Florence, Viale Morgagni, 85, 50134 Florence, Italy. 
Address correspondence to: Dr. Pier Francesco Mannaioni, Department of Preclinical and Clinical Pharmacology, University of Florence, Viale G. Pier-accini, n 6, 50139 Florence, Italy. E-mail: pierfrancesco.mannaioni{at}unifi.it
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, p < 0.05 versus basal.
