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INFLAMMATION AND IMMUNOPHARMACOLOGY
Department of Anesthesiology and Intensive Care Medicine (B), Medical University of Vienna, Vienna, Austria
Received February 7, 2005; accepted July 27, 2005.
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Abstract |
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) and endogenous cannabinoid ligands on various immune functions, mainly in lymphocytes and macrophages.
Suppression of natural killer cell cytotoxicity (Massi et al., 2000), B-lymphocyte activity (Klein et al., 1985), and impairment of macrophage functions (Lopez-Cepero et al., 1986; Baldwin et al., 1997) have been described as well as an enhancement of oxygen radical production in alveolar macrophages (Sarafian et al., 1999) and an increased B-cell proliferation response (Derocq et al., 1995). Some authors also found an altered cytokine production with a shift from T-helper cell 1 to T-helper cell 2 cytokines and an impairment of macrophage/T-cell cooperation induced by certain cannabinoids (Klein et al., 1991, 1998).
To date, cannabinoids are considered to act mainly as immunosuppressive agents in animals and humans because they are potent inhibitors of the adenylate cyclase activity and thus may alter leukocyte functions by reduction of intracellular cAMP levels (Slipetz et al., 1995; Schatz et al., 1997). Reduced intracellular cAMP levels in polymorphonuclear neutrophils have been shown to increase chemotaxis, lysosomal enzyme release, and respiratory burst reaction (Wright et al., 1990). Therefore Gi-coupled receptors such as CB2 would be expected to enhance rather than inhibit neutrophil function. However, not all effects elicited by cannabinoid receptor activation can be explained by cAMP-dependent mechanisms, and most in vitro studies were performed with either isolated cells from animals or transformed cell lines expressing the CB2 receptor. The data reported from these in vitro studies are often unequivocal and difficult to interpret depending on cell type, animal species, cannabinoid compound, concentration, and cellular environment.
Even from in vitro and animal studies, only sparse data exist on the influence of cannabinoids on the major leukocyte population of polymorphonuclear neutrophils (PMNs), although these phagocytes are the first line defense against bacterial and fungal infections. Two independent previous reports showed a suppression of the oxygen radical production of isolated human PMNs at high micromolar concentrations of the natural cannabinoid 
9-tetrahydrocannabinol (THC) and the synthetic THC analog CP55 940 in vitro (Djeu et al., 1991; Kraft et al., 2004). In both studies, the concentrations necessary for this suppression were far above the range that can be reached in vivo, and no data are available on cannabinoid effects under more physiological conditions, i.e., in whole blood. Since dibenzopyrane cannabinoids such as THC (Marinol) or nabilone (Cesamet) are therapeutically used in immunocompromised HIV and cancer patients as antiemetics and to improve appetite, a potential impairment of the phagocytic and oxidative microbicidal activity of human PMNs would be clinically relevant and the conditions of its appearance should be known in more detail.
Therefore, the objective of the present study was to investigate the effects of relevant concentrations of the synthetic THC-analog CP55 940, the endogenous cannabinoid anandamide, and its more stable derivative methanandamide on the respiratory burst of human PMNs in the whole-blood milieu.
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Materials and Methods |
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9-THC, the endogenous eicosanoid compound N-arachidonylethanolamide (anandamide; AEA), and its nonhydrolyzable and thus more stable derivative methanandamide (MethAEA) (Devane et al., 1992; Martin et al., 1999). CP55 940 (Tocris Cookson Inc., Ellisville, MO) and the specific CB2-antagonist SR144 528 (kindly provided by SANOFI Research Center, Montpellier, France) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) and further diluted with phosphate-buffered saline (PBS; Invitrogen, Carlsbad, CA) to the final working concentrations. AEA (Cayman Chemical, Ann Arbor, MI) and MethAEA (Cayman Chemical) were dissolved in ethanol and also diluted with PBS. The sterile solutions were always freshly prepared for each experiment, and the final DMSO or ethanol concentration was 0.001% (v/v) in each test.
Inhibitors of the Cyclooxygenase and Lipoxygenase Enzymes
To characterize the cannabinoid-induced mechanisms and the potential humoral mediators involved, three different inhibitors of cyclooxygenase (COX) or lipoxygenase (LOX) enzymes were used: meclofenamic acid (Sigma-Aldrich), an inhibitor of COX with some LOX-inhibitory effects at higher concentrations (IC50 = 47 µM) (Conroy et al., 1991; Streefkerk et al., 2003); flurbiprofen (Aldrich Chemical Co., Milwaukee, WI), a COX inhibitor without strong suppressive effects on the neutrophil respiratory burst (Parij et al., 1998); and MK886 (kindly provided by Merck Frosst, Canada), an inhibitor of the 5-lipoxygenase activator protein (Daniels et al., 1998). The three inhibitors were dissolved in DMSO and diluted with PBS to the respective working concentrations. The final concentrations in the tests were 50 and 100 µM for meclofenamic acid (Ramos et al., 1994), 10 and 25 µM for flurbiprofen, and 4 and 40 µM for MK886, as previously described by Daniels et al. (1998).
In Vitro Assessment of the Respiratory Burst Reaction in Human Whole-Blood PMNs
After approval by our institutional ethics committee, heparinized venous blood obtained from informed and consenting healthy volunteers (six males, two females, median age 36 years) was incubated with CP55 940 at logarithmic concentration steps from 10-11 to 10-4 M. After incubation, the respiratory burst reaction of the whole-blood PMNs was determined using the commercially available Bursttest (Orpegen Inc., Heidelberg, Germany) as described by Rothe et al. (1998). The synthetic PMN stimulant N-formyl-methionyl-leucylphenylalanine (fMLP; final concentration 10-7 M) or plain buffer were added to 100-µl triplicate aliquots of the whole-blood samples to detect potential stimulatory effects, and the two strong and almost maximum stimulants, the phorbolester PMA (final concentration, 8.1 x 10-7 M) and a suspension of Escherichia coli (109 bacteria/ml), were used for detection of a potential suppressive effect. The burst reaction was determined by the conversion of 123-dihydrorhodamine to the fluorescent dye rhodamine in the cytoplasm of activated PMNs. Rhodamine fluorescence was measured using a FACSCalibur (BD Biosciences, San Jose, CA) flow cytometer with a 488-nm argon laser as previously described (Kraft et al., 2004). Other blood cells were excluded from analysis by a live gate on the PMN cluster in the SSC/FSC dot plot. For each sample, 10,000 events were acquired and aggregation artifacts or cell detritus were detected by the addition of 200 µl of propidium iodide solution (125 mg/ml) after lysing the erythrocytes and washing (live gate on the Fl2 histogram). Data were analyzed with CellQuest software (BD Biosciences), and the mean cellular fluorescence (Fl1), which is proportional to the amount of produced oxygen radicals, as well as the percentage of stimulated rhodamine positive cells were determined to assess the activation of the oxidative burst reaction in whole-blood PMNs (Kraft et al., 2004).
Discrimination of Direct Cellular from Humorally Mediated Cannabinoid Effects
Direct Cellular Effects on PMNs. To further characterize the role of direct cannabinoid effects on PMNs, heparinized venous blood from each healthy donor was divided into two aliquots: one for the whole-blood incubation with the respective cannabinoids as already described above and the other aliquot for the separation of PMNs as described below (Deusch et al., 2003; Kraft et al., 2004). In brief, from the plasma supernatant obtained by Ficoll-Hypaque (Pfizer, Inc., New York, NY) sedimentation, PMNs were separated by centrifugation (20°C, 25 min, 250g) through a two-step Percoll (Pfizer, Inc.) density gradient (62 and 73% v/v). Separated PMNs (2.5 x 106 cells/ml) were resuspended in RPMI 1640 medium supplemented with 5% fetal calf serum and maintained at 37°C. Cell viability was monitored by trypan blue exclusion (>95%), and PMN enrichment was verified by differential count (>95%).
Aliquots of the remaining whole-blood sample from the same donor were incubated with vehicle as controls or the respective cannabinoid concentrations. After centrifugation (20°C, 10 min, 250g), the supernatants were removed and separated into high-molecular mass (>5000 Da) and a low-molecular mass (<5000 Da) fractions by centrifugation through a molecular pore filter system (Centrisart I; Sartorius Inc., Göttingen, Germany) with a cut-off threshold of 5000 Da. Aliquots of the isolated PMNs were incubated (30 min, 37°C) either with the cannabinoid dilution alone or with the respective lowand high-molecular plasma fractions derived from the whole-blood incubation experiments; thereafter, the PMN respiratory burst reaction was examined as described before.
COX- or LOX-Dependent Mediators. The unseparated whole-blood samples were preincubated with meclofenamic acid, flurbiprofen, or MK886 before adding CP55 940 (0.1 and 1.0 nM). Thereafter, the plasma supernatants were obtained by centrifugation (20°C, 10 min, 250g) as described above, and the freshly isolated PMNs from the same donor were incubated with the supernatants (30 min, 37°C) followed by the Bursttest, as described in detail before.
Statistical Analysis. Data were analyzed by means of Jandel Sigma Stat 2.0 software for Windows (SPSS Inc., Chicago, IL). Unless otherwise indicated, results are expressed as a percentage of the control measurements with vehicle alone. For multiple comparisons, one-way ANOVA followed by Bonferroni's post hoc test was used for normally ANOVA-on ranks followed by Dunn's post hoc test for non-normally, distributed values, respectively. Where appropriate, paired Student's t test was applied. P < 0.05 was considered significant.
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). Because of the CB2R dissociation constants for the cannabinoids studied, the nanomolar concentrations were tested because they are considered more relevant to determine CB receptor-mediated effects. The oxygen radical production of fMLP-stimulated whole-blood PMNs was activated at 10-10 and 10-9 M CP55 940, starting after a 90-min incubation period and reaching a maximum after a 120- to 180-min duration of cannabinoid exposure (Fig. 1).
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In contrast to the experiments with the weak stimulus fMLP, the burst enhancement with E. coli was so strong that an additional stimulation with CP55 940 between 0.1 and 1 nM did not reach statistical significance. As expected, PMA produced a maximum stimulation that could not be enhanced by CP55 940 (Fig. 3).
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To rule out an exclusive CP55 940-specific effect, the endocannabinoid AEA and its stable derivative MethAEA were also investigated in our experimental setting. Although anandamide, which is rapidly degraded by fatty acid amido hydrolase in cell-containing media, had neither a stimulatory nor a suppressive effect (data not shown), its nonhydrolyzable derivative MethAEA showed an activation of resting (Fig. 4) and fMLP-stimulated whole-blood PMNs similar to CP55 940 and this effect was also CB2R-dependent as shown by its antagonism with SR144 528 (Fig. 4).
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When the supernatants were separated into a high-molecular mass fraction expected to contain cytokines and a low-molecular mass fraction containing the smaller mediator molecules, such as prostaglandins and leukotrienes, isolated PMNs showed a significant stimulation only after incubation (30 min) with the low-molecular mass fraction but not with the high-molecular mass fraction of the cannabinoid-primed plasma supernatant (Fig. 6).
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The results with the COX inhibitor flurbiprofen (10 and 25 µM, data not shown) were similar to those obtained with meclofenamic acid. The preincubation with flurbiprofen also produced a dose-dependent and significant inhibition of the PMN burst activation mediated by the CP55 940-primed plasma. This effect could be observed in both the resting and fMLP-stimulated PMNs for the percentage of rhodamine-positive cells, as well as for the oxygen radical generation. The preincubation with MK886 (4 and 40 µM), an inhibitor of the 5-lipoxygenase activator protein, did not significantly inhibit the CP55 940-induced stimulation at 10-10 (Tables 2 and 3) and 10-9 M (data not shown).
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Discussion |
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) and 9-THC (Djeu et al., 1991) on isolated human PMNs, this stimulation occurred at much lower and pharmacologically more relevant concentrations of the cannabinoid agents and showed a bell-shaped dose-response relationship with a maximum stimulation between 0.1 and 1.0 nM. Although the 106-fold concentration range of CP55 940 covered more than only the pharmacologically relevant concentrations, those lower than 0.01 nM were not tested; thus, the dose-response relationship was not determined below half-maximum stimulation. However, at high CP55 940 concentrations, the decline in stimulatory activity probably reflects the counteracting receptor-independent, direct inhibitory effect of CP55 940 on PMNs that has been shown to occur in isolated PMNs at similar concentrations. The slight statistically not significant suppression of the respiratory burst reaction of fMLP-treated PMNs at concentrations above 100 nM also supports this interpretation and confirms data from the literature, reporting a concentration-dependent appearance of receptor-mediated versus receptor-independent cannabinoid actions on immune cells. Although the suppressive effect on isolated PMNs was neither CB1R nor CB2R dependent, the stimulatory activity in whole-blood PMNs could be completely blocked by coincubation with the specific CB2R-antagonist SR144 528. In addition, the subnanomolar and low nanomolar concentrations of CP55 940 and the even lower concentrations of MethAEA sufficient to enhance the respiratory burst of resting as well as fMLP-stimulated whole-blood PMNs are consistent with their respective Ki values for CB2 receptors (Rinaldi-Carmona et al., 1998; Howlett et al., 2002).
Together with the SR144 528 antagonism, these findings indicate an involvement of CB2R-mediated mechanisms. Since human PMNs isolated from the blood of healthy individuals lack functional CB2R (Deusch et al., 2003), the failure to detect any direct stimulatory effect of the two cannabinoids on isolated PMNs in the present study confirms our previous results and fits the impression that, although mRNA for CB2R was found by reverse transcription-polymerase chain reaction in PMNs (Bouaboula et al., 1993; Galiegue et al., 1995), human circulating PMNs are not a direct target for cannabinoid actions. Instead, they may be indirectly influenced by the interactions of the cannabinoid agents with other blood cells. The present results with unfractioned and fractioned cell-free plasma supernatants from whole blood exposed to cannabinoids argue for a humoral mechanism that is clearly dependent on a CB2R activation of cannabinoid-sensitive blood cells, presumably macrophages or other peripheral mononuclear leukocytes. The fact that the cannabinoid-induced burst stimulation in whole-blood PMNs did not show a rapid onset but started slowly after an at least 90-min incubation gives further evidence for such an indirect, mediator-dependent mechanism.
Previous reports demonstrated the release of arachidonic acid (Diaz et al., 1994) and the modulation of cytokine production of mononuclear leukocytes (Zhu et al., 1994; Klein et al., 2003) by the marijuana cannabinoid 9-THC. Both mediator pathways are known to be physiological and patho-physiological activators of PMNs, but the fact that in the present study the stimulating activity was exclusively found in the low-molecular mass fraction of the plasma incubation supernatants strongly argues in favor of an the involvement of arachidonic acid or its metabolites. Interactions between prostanoid metabolism and cannabinoids have already been described by various investigators (Burstein et al., 1988; Perez-Reyes et al., 1991) and illustrate the close relationship between arachidonic acid metabolism and endogenous cannabinoid ligands (Edgemond et al., 1998; Pestonjamasp and Burstein, 1998). The activity of COX enzymes and the release of prostaglandin E2 and arachidonic acid were stimulated by cannabinoids in astrocytes (Shivachar et al., 1996), cortical slices (Reichmann et al., 1987), and also lymphocytes (Audette and Burstein 1990). Diaz et al. (1994) demonstrated that THC increased the production of leukotriene B4 and 12-hydroxyeicosatetraenoic acid from mononuclear blood cells. Arachidonic acid and leukotriene B4 are known to be potent chemoattractants, activating PMN migration and oxygen radical generation (Liu et al., 2003). Thus, arachidonic acid and its metabolites are promising candidates for a potential involvement in the observed indirect, low-molecular mass mediator-dependent stimulatory effect of cannabinoids on whole-blood PMNs.
The results with the COX and LOX inhibitors, respectively, suggest mainly the involvement of COX-dependent pathways induced by a CB2R-evoked interaction of the cannabinoids with blood cells others than PMNs. These findings further support the idea of a stimulation of eicosanoid synthesis by cannabinoids as suggested in former investigations by Hunter and Burstein (1997).
Although the short-living, rapidly hydrolyzed endogenous cannabinoid AEA (Di Marzo et al., 1999) had no stimulatory effect on the burst reaction of whole-blood PMNs, its stable, nonhydrolyzable derivative MethAEA enhanced the respiratory burst of PMNs in whole blood, similar to the synthetic 9-THC analog CP55 940. Our observations confirm the CP55 940 data by using a second completely different CB2R ligand and suggest a potential physiological role of endocannabinoids as indirect regulators of PMN activity in humans by means of the COX- or LOX-dependent arachidonic acid pathways in other blood cells. Macrophages (Diaz et al., 1994) or mast cells (Samson et al., 2003) are possible candidates for the release of arachidonic acid into whole blood after cannabinoid incubation. Interestingly enough, the exposure to marijuana smoke was recently reported to increase the oxygen radical production from alveolar macrophages in humans, resulting in oxidative stress and inflammation (Baldwin et al., 1997).
Although the endogenous cannabinoid, 2-arachidonylglycerol (2-AG), also acts on CB2R and is supposed to play a role in immunomodulation (Sugiura and Waku, 2000), experiments with 2-AG would have been hampered by its significant instability in cell culture media (Rouzer et al., 2002). Even under cell-free conditions, 2-AG rapidly rearranges to 1- or 3-arachidonylglycerol in a first order process with a half-life of only 2.3 min in RPMI 1640 medium containing 10% fetal calf serum. Because AEA resembles 2-AG in its affinity to the CB2R (Gonsiorek et al., 2000) and, what is more important from a practical point of view, has the stable derivative MethAEA available, only AEA and MethAEA were used as endocannabinoid substances in our experiments.
In conclusion, our results suggest that human circulating PMNs are not a direct cellular target of endogenous or exogenous cannabinoids but are nevertheless strongly activated by a CB2R-evoked COX-dependent mediator pathway induced by cannabinoid interactions with other blood cells. Thus, there is no evidence for a potential cannabinoid-induced suppression of PMN functions in healthy human individuals, but in contrast, even an enhancement of oxidative burst activity is to be expected.
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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: CB2R, cannabinoid receptor; PMN, polymorphonuclear neutrophil; THC, 9-tetrahydrocannabinol; CP55 940, (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; AEA, anandamide; MethAEA, methanandamide; SR144 528, 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; PBS, phosphate-buffered saline; COX, cyclooxygenase; LOX, lipoxygenase; MK886, 3-[1-(p-chlorobenzyl)-5(isopropyl)-3-t-butylthioindol-2-yl]-2,2-dimethylpropanoic acid,Na; fMLP, N-formyl-methionyl-leucyl-phenylalanine; PMA, phorbol 12-myristate 13-acetate; ANOVA, analysis of variance; 2-AG, 2-arachidonoylglycerol.
Address correspondence to: Dr. Birgit Kraft, Department of Anesthesiology and Intensive Care Medicine (B), Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: birgit.kraft{at}meduniwien.ac.at
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