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Originally published online as doi:10.1189/jlb.0304184 on July 16, 2004

Published online before print July 16, 2004
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(Journal of Leukocyte Biology. 2004;76:854-861.)
© 2004 by Society for Leukocyte Biology

The THC-induced suppression of Th1 polarization in response to Legionella pneumophila infection is not mediated by increases in corticosterone and PGE2

Catherine A. Newton*, Tangying Lu*, Stanley J. Nazian{dagger}, Izabella Perkins*, Herman Friedman* and Thomas W. Klein*,1

* Departments of Medical Microbiology and Immunology and
{dagger} Physiology and Biophysics, University of South Florida, College of Medicine, Tampa

1Correspondence: University of South Florida, College of Medicine, MDC Box 10, 12901 Bruce Downs Blvd., Tampa, FL 33612. E-mail: tklein{at}hsc.usf.edu


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ABSTRACT
 
T helper cell type 1 (Th1)-polarizing cytokines are induced by Legionella pneumophila infection and are suppressed by pretreatment with marijuana cannabinoids (CB). Glucocorticoids and prostaglandin E2(PGE2) are also reported to suppress Th1 polarization and are induced by {Delta}9-tetrahydrocannabinol (THC), so their role in the suppression of polarizing cytokines was examined. Injection of L. pneumophila or THC alone into BALB/c mice induced a rapid and transient rise in serum corticosterone (CS), and the injection of both agents significantly augmented the CS response, demonstrating that THC increased CS in Legionella-infected mice. Pretreatment with the CB receptor 1 (CB1) antagonist SR141716A had no effect on the THC-induced CS response, but CB2 antagonist (SR144528) treatment increased the CS response. To see if increased CS contributed to the down-regulation of Th1 cytokines, mice were pretreated with the steroid antagonist RU486 before THC injection and Legionella infection. The results showed that RU486 did not attenuate the THC-induced suppression of serum interleukin (IL)-12 or interferon-{gamma} (IFN-{gamma}). In addition to CS, THC injection increased urinary PGE2 metabolites, and the CB1 antagonist attenuated this increase. Although L. pneumophila infection increased urinary PGE2, THC pretreatment did not enhance this response; in addition, treatment with the cyclooxygenase inhibitor, indomethacin, did not block the THC-induced suppression of IL-12 and IFN-{gamma}. These results suggest that the elevation of CS and PGE2 does not account for the THC-induced attenuation of the Th1 cytokine response, and it is concluded that other suppressive mediators are induced by THC or that the drug acts directly on immune cells to suppress cytokine production.

Key Words: cannabinoids • Th2 cells • IL-12 • prostaglandins


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INTRODUCTION
 
The major psychoactive component of marijuana, {Delta}9-tetrahydrocannabinol (THC), has various immunomodulatory properties, some of which are mediated through cannabinoid receptors (CBR) [1 2 3 ]. These receptors contain seven transmembrane domains and are G protein-coupled receptors. Two have been identified: CB1 and CB2 [4 ]. CB1 is expressed in the brain and in some peripheral tissues such as spleen, heart, kidney, small intestine, testis, and ovary. CB2 is predominately expressed in the periphery and on immune cells. Experiments with CBR-deficient mice indicate that CB1 mediates the behavioral effect of THC [5 , 6 ], and CB2 is important in at least some immunomodulatory functions of THC [7 ].

Legionella pneumophila, an intracellular bacterium and causative agent of Legionnaires’ disease and Pontiac fever, causes an acute, febrile disease in BALB/c mice. In addition, THC injection increases mortality to a challenge infection [8 , 9 ] by reducing the development of protective T helper cell type 1 (Th1) responses. In general, the development of Th1 immunity depends on the production of polarizing cytokines such as interleukin (IL)-12 by antigen-presenting cells as well as the up-regulation of IL-12 receptor ß 2 (IL-12Rß2) [10 , 11 ], which mediates the production of interferon-{gamma} (IFN-{gamma}) [12 ]. The influence of IL-12 is so strong that Th2-dominant mouse strains such as BALB/c can be converted to a Th1 phenotype by treatments that increase IL-12 [13 , 14 ], and in support of this, we have shown that Legionella infection of these mice induces Th1 immunity and is a potent inducer of IL-12. THC treatment prior to infection reduces Th1-biasing responses such as levels of IL-12, IFN-{gamma}, and splenic IL-12Rß2 mRNA [9 ]. Furthermore, antagonists for CB1 (SR141716A; SR1) and CB2 (SR144528; SR2) attenuate this suppression, suggesting that both receptors are involved in the drug effect. A similar THC-induced suppression of Th1 activity has also been reported in marijuana-smoking humans [15 ] as well as in other experimental systems involving human [16 ] and mouse [17 18 19 ] immune cells.

Th1 polarization is inhibited by a number of factors such as the elevation of IL-4 [10 ], activation of transcription factors such as GATA3 [20 , 21 ] and nuclear factor-{kappa}B [22 ], Gi protein signaling [23 ], and increases in prostaglandins [24 , 25 ] and glucocorticoids [24 , 26 , 27 ]. In our L. pneumophila model, increased IL-4 was observed [28 ]; however, this increase was not potently linked to the THC effect, as IL-12 and IFN-{gamma} activities were still suppressed in THC-treated, IL-4-deficient mice [9 ], and others have shown [29 ] that increased IL-4 following infection is not always associated with suppression of Th1 immunity. In addition to IL-4, glucocorticoids and prostaglandins are increased following administration of THC into rodents and therefore, could account for the drug effect on Th1 cytokines. For example, oral administration of THC in rats and mice was shown to enhance brain levels of prostaglandin E2(PGE2) [30 ], the injection of THC into the brains of rats elevated the serum level of adrenocorticotropin and corticosterone (CS) [31 ], and intraperitoneal (i.p.) injection induced corticotrophin and CS [32 ]. In addition to THC effects on stress hormones, CBR have been observed in the hypothalamus [33 ] and pituitary gland [34 ], further suggesting a link between THC and the stress response. Finally, in addition to suppression of Th1 responses, corticosteriods and prostaglandins have been observed to promote the shift from Th1 to Th2 [25 , 35 , 36 ], suggesting their involvement in Th1 modulation.

The objective of the current study was to determine if THC-induced increases in CS and PGE2 accounted for the drug suppression of the Th1-biasing cytokines IL-12 and IFN-{gamma}. We observed that L. pneumophila infection increased serum CS and that pretreatment with THC augmented this response. However, the steroid antagonist RU486 did not fully attenuate the THC-induced suppression of Th1-biasing cytokines. Regarding prostaglandins, THC treatment increased urinary PGE2, and this was attenuated by prior treatment with the CB1 antagonist. However, although L. pneumophila infection increased urinary PGE2, THC pretreatment did not increase the eicosanoid further, and indomethacin treatment did not attenuate the THC-induced suppression of Th1 cytokines. These results suggest that although THC treatment significantly augments the mobilization of corticosteroids and prostaglandins, these increased levels do not account for the drug-induced suppression of Th1 activity in the model.


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MATERIALS AND METHODS
 
Bacteria and drugs
L. pneumophila M124, a serogroup 1 isolate from a case of Legionnaires’ disease, was grown on buffered charcoal yeast extract (BD-Difco, Sparks, MD) plates for 48 h from a passage 3 stock maintained at –80°C. The Research Technology Branch of the National Institute on Drug Abuse provided SR1 (CB1 antagonist), SR2 (CB2 antagonist), and THC. All drugs were endotoxin-free, as judged by analysis with the QC L-1000 (Cambrex, Baltimore, MD), which has a sensitivity of 0.1 endotoxin units/ml. The antagonists and THC were diluted first in dimethyl sulfoxide (DMSO) to 50 mg/ml and then in mouse serum to 1 or 2 mg/ml. The steroid receptor antagonist RU486 (mifepristone) and PEG400 were obtained from Sigma Chemical Co. (St. Louis, MO). This compound has a high affinity for the progesterone and glucocorticosteroid receptors [37 ] and when administered to animals, has been shown to block the biological activity of these two steroid hormones.

Animals
Female BALB/cByJ (Jackson Labs, Bar Harbor, ME) were used at 8–10 weeks of age. The mice were housed and cared for in the animal facility of University of South Florida Heath Sciences Center (Tampa), which is accredited by the American Association for Accreditation of Laboratory Animal Care. Mice were infected through tail vein [intravenous (i.v.)] injections of L. pneumophila (7x106 per mouse). When given, mice were injected i.v. with THC (8 mg/kg; 200 µg/mouse) 18 h prior to infection. For the antagonist studies, the SR compounds (4 mg/kg; 100 µg/mouse) were injected i.v. 30 min prior to the THC treatment. We prefer to use the i.v. route of injection, as it delivers drugs to the blood more rapidly and efficiently than i.p. injection. This direct blood delivery closely mimics marijuana smoking, in that full catalepsy occurs in the mice in less than 5 min, and urinary drug metabolite output mimics the human condition in terms of metabolites produced and kinetics (unpublished observation). It should also be noted that i.v. injection has recently been shown to be an efficient delivery method of THC in humans and animals [38 , 39 ]. For PGE2 and CS analysis, urine and blood (collected by cardiac puncture) were taken at the indicated times after infection. For glucocorticoid receptor antagonist studies, RU486 was dissolved in PEG400 (5 mg/ml) and injected subcutaneous in the right and left flanks of the mouse (50 µl/side) 1 h prior to THC pretreatment or L. pneumophila infection [40 , 41 ]. To investigate PGE2 inhibition, indomethacin was dissolved in 0.1 M sodium carbonate, further diluted in pyrogen-free saline (2 mg/ml), and injected i.v. in 0.125 ml, 1 h prior to THC treatment.

CS Radioimmunoassay (RIA)
CS levels in the sera were determined using a double-antibody RIA kit from ICN Biomedicals (Costa Mesa, CA). Samples from three to four experiments were run in the same assay to avoid interassay variations. The intra-assay coefficients of variation averaged 7.0%. Approximately 5 ng/ml was the minimum detectable concentration.

Cytokine enzyme-linked immunosorbent assay (ELISA)
Sera were diluted 1:2 and tested for IFN-{gamma} and IL-12p40/p70 activity by sandwich ELISAs using antibody pairs from BD-PharMingen (San Diego, CA) following the protocols described previously [9 ]. The plates were read at 450 nm on an Emax microplate reader (Molecular Devices, Mento Park, CA), and units were calculated from the standard curve, performed for each plate. The low-end sensitivity was 200 pg/ml for each cytokine.

Bicyclo PGE2 enzyme immunoassay (EIA)
A competitive ACETM EIA for bicyclo PGE2 (Cayman, Ann Arbor, MI) was used to determine PGE2 levels. Collected urine was incubated in 1 M carbonate buffer overnight to convert all of the unstable metabolites of PGE2 to the stable bicyclo PGE2 derivative. The urine was neutralized and diluted to 1:75–100 for measurements. The sensitivity of the EIA was between 3 and 5 pg/ml.

Statistical analysis
Data were analyzed by one-way analysis for variance with Dunnett’s test for comparing groups, using SigmaStat (Jandel Scientific, San Rafael, CA).


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RESULTS
 
THC injection and/or L. pneumophila infection increases serum CS
Previous studies demonstrated that CS increased in rats following THC injection [31 , 32 ]. Initially, we tested for a similar response in mice. Animals were injected with THC, blood collected at various times following injection, and the serum examined by RIA for CS. Figure 1 shows that saline and DMSO injections increased CS over normal animals, probably as a result of the stress of injection and that THC injection further increased the steroid concentration. The peak increase occurred within 3 h following THC injection, returning to normal levels by 18 h (Fig. 1) . A previous report showed that the THC-induced increase in CS in rats was attenuated by prior treatment with the CB1 antagonist [42 ]. To determine if the antagonist had a similar effect in our model, mice were injected with SR1 (CB1 antagonist) 30 min prior to THC injection, and the serum was collected 3 h after THC treatment. The results showed (Fig. 1) that unlike in the previous report, SR1 pretreatment did not attenuate the THC-induced increase in serum CS. Regarding the SR2 antagonist, the data showed that it increased the CS response to THC treatment instead of attenuating it (Fig. 1) . These data suggested that CB1 was not solely involved in the induction of the CS response in our model; however, CB2 might have an attenuating role on CS mobilization, as the steroid response is enhanced in the presence of THC and SR2.



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  		Figure 1. THC treatment increases serum CS. Groups of mice were i.v. injected with saline (S) or DMSO (D; 0.05%) or were not injected (N), and sera were collected at 3 h post-treatment for CS analysis. Other groups were injected with THC only (8 mg/kg), and the sera were collected at various times up to 18 h for analysis. In other groups, antagonists for the CB1 receptor (SR1; 4 mg/kg) or the CB2 receptor (SR2; 4 mg/kg) were injected 30 min prior to THC injection or prior to saline injection, and sera were taken from these animals 3 h postinjection. Serum CS was measured by RIA as described in Materials and Methods. The data represent the mean of three experiments ± SEM. *,DMSO and Sham significantly different from normal. **, Significantly different from DMSO group. ***, Significantly different from THC-only group; P < 0.05.

CS levels were also measured in mice infected with L. pneumophila (Fig. 2 ). These animals displayed a surprisingly strong elevation of serum CS that remained elevated through 5 h. Furthermore, THC injected 18 h prior to infection further augmented this increase at 1, 2, and 3 h (Fig. 2) . These results showed that THC injection increased serum CS and that THC increased the L. pneumophila-induced increase in the steroid response, suggesting that CB pretreatment primed the animal for an enhanced steroid response following infection.



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  		Figure 2. L. pneumophila (Lp; 1x107/ms) elevates CS levels, and THC (THC/Lp; 8 mg/kg) pretreatment augments this increase. Mice were injected with saline (Sham), infected with Lp, or injected with THC followed by Lp infection, and sera were collected. The CS levels were determined by RIA. The data represent the mean ± SEM of three to five experiments with a total of five to 18 mice. *, THC/Lp significantly different from Lp only. #, Lp significantly different from Sham; P < 0.05.

No involvement of CBR in the combined treatment with THC and L. pneumophila
The above results showed that the THC-induced increase in CS was CB1-independent but possibly attenuated by CB2 activity. To determine if the combined effect of THC injection and L. pneumophila infection had a receptor-mediated component, mice were injected with SR1 or SR2 30 min prior to THC or saline treatment. Animals were then infected 18 h later and bled at 2 h postinfection, and the level of serum CS was determined. Figure 3 showed that neither antagonist modulated the THC effect on CS elevation measured after Legionella infection. However, it is interesting that the SR compounds slightly but significantly caused an increase in CS when measured 20 h after their administration and 2 h after Legionella infection. These results suggested that either CBR is not involved in the induction of the CS response following cotreatment with THC and Legionella or that as THC can bind to both receptors, one receptor compensates for the other under conditions of single antagonist treatment. The results with SR treatment and infection only suggested that Legionella induced endocannabinoids, which have a suppressive effect on CS.



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  		Figure 3. SR1 and SR2 effects on CS increase in response to THC and L. pneumophila (Lp) treatment. Mice were pretreated with SR1 (4 mg/kg) or SR2 (4 mg/kg) 30 min prior to THC (8 mg/kg) or saline injection. The mice were then infected with L. pneumophila 18 h later and bled 2 h postinfection for CS measurements. Data are the means ± SEM from three experiments. *, Significantly different from the Lp group. #, Significantly different from the Lp group; P < 0.05.

RU486 does not fully attenuate THC-induced suppression of the Th1-polarizing cytokines
The previous experiments suggested that THC injection significantly increased CS within hours following L. pneumophila infection at a time when Th1-polarizing cytokines are being mobilized. To determine if the CS increase suppressed in some way the mobilization of IL-12 and IFN-{gamma}, mice were pretreated with the steroid inhibitor RU486 and then tested for the THC effect. RU486 was injected 1 h before THC injection or 1 h before L. pneumophila infection, and serum was collected at 8 h following infection and analyzed for IL-12 and IFN-{gamma} by ELISA. Eight hours was selected, as previous studies showed this to be the optimal time for cytokine enhancement following THC and L. pneumophila infection [9 ]. Figure 4 showed that THC injection, 18 h prior to infection, suppressed the normal increase in serum IL-12 and IFN-{gamma}. Pretreatment with RU486 before THC injection or before L. pneumophila infection did not attenuate the THC inhibition of IL-12 and IFN-{gamma} (Fig. 4A and 4B) ; however, the steroid inhibitor did increase the overall production of IFN-{gamma} in all groups (Fig. 4B) . These results showed that the steroid antagonist had no effect on THC-induced suppression of IL-12 and IFN-{gamma} production in response to Legionella infection. The enhancing effect of RU486 on IFN-{gamma} production has been reported previously and is a result of an inhibition of the glucocorticoid suppression of the phosphorylation of signal transducer and activator of transcription (STAT)4 [43 ].



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  		Figure 4. RU486, a glucocorticoid receptor antagonist, has no effect on THC-induced suppression of IL-12 (A) and IFN-{gamma} (B) serum levels. Groups of mice (untreated) were L. pneumophila (Lp)-infected only or THC-injected and Lp-infected as before. In addition, groups were treated with RU486 (20 mg/kg) 1 h prior to THC pretreatment or 1 h prior to Lp infection, and sera were collected at 8 h postinfection for IL-12 and IFN-{gamma} analysis. *, Significantly different from Lp only group. +, Significantly different from untreated Lp group. #, Significantly different from untreated THC/Lp group. Data are presented as the mean ± SEM for three experiments; P< 0.05.

THC induces an increase in PGE2 by a CB1-mediated mechanism
Prostaglandins have also been shown to suppress Th1 activity, and these markers of the systemic inflammatory response of the host to infectious stimuli can be monitored by various methods including the urinary excretion of PGE2 metabolites [44 ]. Therefore, to monitor systemic PGE2 mobilization following THC treatment, urine was collected at different time-points after THC injection. A competitive bicyclo PGE2 ELISA assay was used to determine the level of excreted PGE2 metabolites. THC injection produced a rapid and transient elevation in PGE2 excretion (Fig. 5A ), and the eicosanoid increased within 0.5 h following THC injection and declined by 2 h before returning to normal by the time of L. pneumophila infection (18 h later). To determine the role of CBR, mice were pretreated with SR1 or SR2 receptor antagonists 30 min prior to THC or saline treatment. Figure 5B shows that SR1 but not SR2 attenuated the rise at 0.5 h after THC injection, suggesting that the increase was CB1-mediated. Treatment with SR1 or SR2 only had no effect on PGE2 excretion.



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  		Figure 5. THC increases the bicycle PGE2 level in the urine by a CB1-mediated mechanism. Groups of mice were treated with THC (8 mg/kg) or DMSO (D), and urines were collected at various times thereafter (A). In addition, mice were pretreated with SR1 (4 mg/kg) or SR2 (4 mg/kg) 30 min prior to THC or saline treatment, and urines were collected 0.5 h later (B). Urines were analyzed by ELISA, and the data were expressed as the mean ± SEM for three to five experiments. *, Significantly different from the DMSO group; P < 0.05.

THC injection has no effect on PGE2 elevation following Legionella infection
The infection model under study involves the treatment of mice with not only THC but also L. pneumophila. Therefore, urinary PGE2 levels were measured in animals injected with THC followed 18 h later by L. pneumophila infection. Figure 6 shows that infection alone significantly increased urinary PGE2 excretion at 0.5 h and 2 h following infection, and the PGE2 level declined at the latter time-point; however, THC treatment 18 h before did not augment the prostaglandin response over that of infection only, and pretreatment with SR1 and SR2 also had no effect on the prostaglandin excretion pattern. These results suggested that as THC treatment had no effect on PGE2 mobilization over that caused by L. pneumophila infection only, PGE2 mobilization had little effect on Th biasing occurring after infection.



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  		Figure 6. THC injection does not augment the L. pneumophila-induced increase in urinary PGE2. Groups of mice were untreated (Normal) or pretreated with the SR antagonists (4 mg/kg) 30 min prior to being injected with THC (8 mg/kg) or DMSO (0.1%). The treated groups were then infected with L. pneumophila (Lp) 18 h later, and urine was collected at 0.5 h and 2 h after infection. Urines were analyzed by ELISA, and the data were expressed as the mean ± SEM for three to five experiments. *, Significantly different from the Normal group; P < 0.05.

Indomethacin treatment does not attenuate THC-induced suppression of Th1-biasing cytokines
The above studies cast doubt on the causative role of prostaglandins in Th biasing. To study this more directly, mice were pretreated with indomethacin, a nonselective, cyclooxygenase inhibitor, prior to THC injection and L. pneumophila injection. Eight hours following infection, blood serum was collected, and the level of IL-12 and IFN-{gamma} was determined by ELISA. Figure 7 shows that indomethacin treatment did not attenuate the THC-induced suppression of Th1-biasing cytokines. THC equally suppressed IL-12 and IFN-{gamma} levels in the presence or absence of indomethacin. Figure 7C shows that the suppression of the biasing cytokines occurred in spite of the fact that indomethacin treatment inhibited the mobilization of PGE2.



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  		Figure 7. Indomethacin pretreatment has no effect on THC-induced suppression of serum IL-12 and IFN-{gamma}. Mice were treated 1 h prior to THC (8 mg/kg) with indomethacin (10 mg/kg) and infected 18 h later with L. pneumophila (Lp). Sera were collected 8 h after infection, and IL-12 and IFN-{gamma} were measured by ELISA (A and B). Urines were also collected for PGE2 analysis by ELISA (C). Data are expressed as the mean ± SEM of three experiments. *, Significantly different from the Lp-only group; P < 0.05.


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DISCUSSION
 
In this study, we examined the possible roles of glucocorticoids and prostaglandins in THC-induced suppression of Th1 polarization in response to Legionella infection. Initially, mice were i.v.-injected with THC only, and the CS response was measured by RIA. Mice displayed a response similar to that reported in rats [31 ] with a robust increase in serum CS within the first few hours following THC treatment (Fig. 1) . This increase rapidly declined and returned to normal levels by 18 h following injection. However, the THC effect on CS was not attenuated by pretreatment with SR1 (Fig. 1) . This finding was surprising, as previous results showed that SR1 pretreatment attenuated the CS response [42 ]. Furthermore, CB1 receptors have been shown to be involved in the drug-induced suppression by other hormones, such as luteinizing hormone, prolactin, and testosterone [34 , 45 ], and these receptors have also been demonstrated in the hypothalamus and the anterior pituitary [45 ]. However, it is noteworthy that CB1 protein was reported to be most prominent in lactotroph cells and luteinizing hormone-secreting gonadotrophs in the pituitary and less pronounced in corticotroph cells [46 ]. It is also noteworthy that in the rat studies, the antagonist and THC were injected intracerebroventricularly, whereas in our studies, all injections were given i.v. It is also possible that THC is working through a subtype of CB1 that is relatively insensitive to the SR compounds as suggested by others [47 ], or THC injected in the periphery is inducing a second mediator that is inducing CS by a CBR-independent mechanism. Both of these possibilities are testable and thus await further study. In addition to studies with SR1, we examined the effect of SR2 on the THC-induced increase in CS and found that this antagonist enhanced rather than suppressed the CS response. This type of augmented THC effect by SR2, to our knowledge, has not been reported, and the mechanism is unclear at this time. It is possible that THC, injected peripherally, in addition to increasing CS by means of a CB1-independent mechanism, is working through a CB2 mechanism to suppress CS, and we are currently studying this prospect.

We next examined CS levels in mice injected with THC and L. pneumophila. Figure 2 shows that Legionella infection alone significantly increased CS within hours, and this rise was significantly increased by a THC injection 18 h before infection. The way in which THC injection mediates this effect is not clear at this time. It is possible that the pretreatment with THC sensitized the hypothalamic-pituitary-adrenocortical axis so that the subsequent stress of infection resulted in a hyper-responsiveness and outpouring of CS. We also examined the effect of SR antagonists on the combined treatment with THC and Legionella, and neither antagonist was shown to have an effect (Fig. 3) . The effect of SR pretreatment was also examined in mice Legionella infected 18 h later, and surprisingly, both antagonists slightly but significantly increased the CS response 2 h after infection (Fig. 3) . Bacterial endotoxin has been shown to increase the production of the endocannabinoid, anandamide [48 ], which has been shown to increase CS [31 ]. We are currently examining the possibility that Legionella infection induces tissue expression of anandamide and 2-arachidonoyl glycerol, which in turn, could be causing an increase in CS.

As CS was increased in our model in response to THC treatment and as the steroid has been shown to suppress Th1 development [24 , 27 ], we wanted to test the role of CS directly in Th biasing. Mice were treated with the steroid receptor antagonist RU486 before THC treatment or before L. pneumophila infection, and the serum levels of IL-12 and IFN-{gamma} were tested 8 h after infection. Figure 4 showed that RU486 did not interfere with the THC-induced suppression of IL-12, suggesting that this effect was not mediated by CS elevation. A variety of reports has demonstrated that glucocorticoids can suppress IL-12 production in vitro [26 , 49 , 50 ] and that this effect is inhibited by RU486 [26 , 49 ]. However, to our knowledge, this has not been shown in vivo, and our results would suggest that at the whole animal level, the linkage between steroid receptor activation and IL-12 suppression is less certain and that other receptors and/or factors might be involved. The effect of RU486 was also tested on serum IFN-{gamma} levels and was shown to increase its overall production (Fig. 4) . However, THC treatment still suppressed IFN-{gamma} production in the presence of RU486, showing that steroids did not mediate the CB-suppressive effect on cytokine production. The overall increase in IFN-{gamma} production caused by RU486 supports previous findings that glucocorticoids inhibit IL-12-induced STAT4 phosphorylation and IFN-{gamma} production [43 ]. These inhibitor studies strongly suggest that mechanisms other than increasing CS are involved in the THC-induced suppression of IL-12 and IFN-{gamma} production following Legionella infection.

Prostaglandins, especially PGE2, have, for years, been considered immunosuppressive, and this activity has more recently been linked to polarization of Th cells with a biasing toward Th2 cells [24 , 25 , 51 , 52 ] and enhanced susceptibility to infection with intracellular pathogens [53 , 54 ]. Our data showed that a single injection of THC induced a 60% increase in the urinary excretion of PGE2 metabolites within 30 min of injection (Fig. 5A) . This level of increase is in the response range of inflammatory agents such as bacterial lipopolysaccharide and IL-1 [44 ]. Figure 5 also showed that the CB1 antagonist SR1 attenuated excretion of PGE2. Previous work suggested that CB1 receptors are involved in the THC-induced stimulation of arachidonic acid release using an in vitro model system [55 ], and our results support this finding. We also tested the effect of THC in the infection model. The results showed that Legionella infection alone increased urinary PGE2 metabolites (Fig. 6) ; however, pretreatment with THC did not further increase this effect. This suggests that the THC effect on suppression of IL-12 and IFN-{gamma} mobilization is not a result of an increase in prostaglandins, as the level shortly after infection appears to be the same, irrespective of whether the animals are injected with THC. In addition, a role for prostaglandins is unlikely, as indomethacin treatment had no effect on cytokine mobilization (Fig. 7) . Although indomethacin and other similar agents have been shown to modulate cytokine production [54 ], these studies differed from ours in that the indomethacin was given chronically for weeks instead of by a single injection prior to THC treatment. Thus, although prostaglandins are reported to modulate Th1 immunity, the early and transient rise in our studies did not appear to suppress the polarizing cytokines. The current findings are based on the measurement of urinary PGE2 excretion; however, it is possible that the measurement of splenic PGE2 tissue levels might support different conclusions.

In summary, although CS and PGE2 are reported in other studies to bias toward Th2 immunity, they do not appear to do so in our model. It is possible that THC treatment biases toward Th2 immunity by promoting the default pathway [21 ]. This pathway defaults to Th2 immunity under conditions wherein the IL-12 system is suppressed in some way, and the level of GATA3 is allowed to increase. We have observed that THC treatment increases GATA3 [56 ] and decreases IL-12, and therefore, it is possible that the drug, instead of working through mediators such as CS and PGE2, is acting directly on lymphocytes and dendritic cells (DC), disrupting the expression of GATA3 and other key biasing factors. We are currently examining this possibility in DC models.


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ACKNOWLEDGEMENTS
 
This work was supported in part by grants from the National Institutes of Health (DA03646 and AI45169).

Received March 22, 2004; revised June 7, 2004; accepted June 21, 2004.


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REFERENCES
 
    1
  1. Klein, T., Newton, C., Friedman, H. (1998) Cannabinoid receptors and immunity Immunol. Today 19,373-381[CrossRef][Medline]
    2. 2
  2. Klein, T. W., Lane, B., Newton, C. A., Friedman, H. (2000) The cannabinoid system and cytokine network Proc. Soc. Exp. Biol. Med. 225,1-8[Abstract/Free Full Text]
    3. 3
  3. Klein, T. W., Newton, C., Larsen, K., Lu, L., Perkins, I., Nong, L., Friedman, H. (2003) The cannabinoid system and immune modulation J. Leukoc. Biol. 74,486-496[Abstract/Free Full Text]
    4. 4
  4. Pertwee, R. G. (1997) Pharmacology of cannabinoid CB1 and CB2 receptors Pharmacol. Ther. 74,129-180[CrossRef][Medline]
    5. 5
  5. Ledent, C., Valverde, O., Cossu, G., Petitet, F., Aubert, J. F., Beslot, F., Bohme, G. A., Imperato, A., Pedrazzini, T., Roques, B. P., Vassart, G., Fratta, W., Parmentier, M. (1999) Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice Science 283,401-404[Abstract/Free Full Text]
    6. 6
  6. Zimmer, A., Zimmer, A. M., Hohmann, A. G., Herkenham, M., Bonner, T. I. (1999) Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice Proc. Natl. Acad. Sci. USA 96,5780-5785[Abstract/Free Full Text]
    7. 7
  7. Buckley, N. E., McCoy, K. L., Mezey, E., Bonner, T., Zimmer, A., Felder, C. C., Glass, M. (2000) Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB(2) receptor Eur. J. Pharmacol. 396,141-149[CrossRef][Medline]
    8. 8
  8. Newton, C. A., Klein, T. W., Friedman, H. (1994) Secondary immunity to Legionella pneumophila and Th1 activity are suppressed by {delta}-9-tetrahydrocannabinol injection Infect. Immun. 62,4015-4020[Abstract/Free Full Text]
    9. 9
  9. Klein, T. W., Newton, C. A., Nakachi, N., Friedman, H. (2000) {Delta}9-Tetrahydrocannabinol treatment suppresses immunity and early IFN{gamma}, IL-12, and IL-12 receptor ß2 responses to Legionella pneumophila infection J. Immunol. 164,6461-6466[Abstract/Free Full Text]
    10. 10
  10. Szabo, S. J., Dighe, A. S., Gubler, U., Murphy, K. M. (1997) Regulation of the interleukin (IL)-12R B2 subunit expression in developing T helper 1 (Th1) and Th2 cells J. Exp. Med. 185,817-824[Abstract/Free Full Text]
    11. 11
  11. Pulendran, B., Palucka, K., Banchereau, J. (2001) Sensing pathogens and tuning immune responses Science 293,253-256[Abstract/Free Full Text]
    12. 12
  12. Wu, C., Wang, X., Gadina, M., O’Shea, J. J., Presky, D. H., Magram, J. (2000) IL-12 receptor ß 2 (IL-12R ß 2)-deficient mice are defective in IL-12-mediated signaling despite the presence of high affinity IL-12 binding sites J. Immunol. 165,6221-6228[Abstract/Free Full Text]
    13. 13
  13. Scott, P., Eaton, A., Gause, W. C., di Zhou, X., Hondowicz, B. (1996) Early IL-4 production does not predict susceptibility to Leishmania major Exp. Parasitol. 84,178-187[CrossRef][Medline]
    14. 14
  14. Ramirez-Pineda, J. R., Frohlich, A., Berberich, C., Moll, H. (2004) Dendritic cells (DC) activated by CpG DNA ex vivo are potent inducers of host resistance to an intracellular pathogen that is independent of IL-12 derived from the immunizing DC J. Immunol. 172,6281-6289[Abstract/Free Full Text]
    15. 15
  15. Pacifici, R., Zuccaro, P., Pichini, S., Roset, P. N., Poudevida, S., Farre, M., Segura, J., De la Torre, R. (2003) Modulation of the immune system in cannabis users JAMA 289,1929-1931[Free Full Text]
    16. 16
  16. Yuan, M., Kiertscher, S. M., Cheng, Q., Zoumalan, R., Tashkin, D. P., Roth, M. D. (2002) D9-tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells J. Neuroimmunol. 133,124-131[CrossRef][Medline]
    17. 17
  17. Smith, S. R., Terminelli, C., Denhardt, G. (2001) Modulation of cytokine responses in Corynebacterium parvum-primed endotoxemic mice by centrally administered cannabinoid ligands Eur. J. Pharmacol. 425,73-83[CrossRef][Medline]
    18. 18
  18. Zhu, L. X., Sharma, S., Stolina, M., Gardner, B., Roth, M. D., Tashkin, D. P., Dubinett, S. M. (2000) {Delta}-9-Tetrahydrocannabinol inhibits antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway J. Immunol. 165,373-380[Abstract/Free Full Text]
    19. 19
  19. Massi, P., Fuzio, D., Vigano, D., Sacerdote, P., Parolaro, D. (2000) Relative involvement of cannabinoid CB(1) and CB(2) receptors in the {delta}(9)-tetrahydrocannabinol-induced inhibition of natural killer activity Eur. J. Pharmacol. 387,343-347[CrossRef][Medline]
    20. 20
  20. Kuo, C., Leiden, J. (1999) Transcriptional regulation of T lymphocyte development and function Annu. Rev. Immunol. 17,149-187[CrossRef][Medline]
    21. 21
  21. Moser, M., Murphy, K. (2000) Dendritic cell regulation of Th1-Th2 development Nat. Immunol. 1,199-205[CrossRef][Medline]
    22. 22
  22. Das, J., Chen, C-H., Yang, L., Cohn, L., Ray, P., Ray, A. (2001) A critical role for NF-{kappa}B in GATA3 expression and Th2 differentiation in allergic airway inflammation Nat. Immunol. 2,45-50[CrossRef][Medline]
    23. 23
  23. He, J., Gurunathan, S., Iwasaki, A., Ash-Shaheed, B., Kelsall, B. (2000) Primary role for Gi protein signaling in the regulation of interleukin 12 production and the induction of T helper cell type 1 responses J. Exp. Med. 191,1605-1610[Abstract/Free Full Text]
    24. 24
  24. Wu, C-Y., Wang, K., McDyer, J. F., Seder, R. A. (1998) Prostaglandin E2 and dexamethasone inhibit IL-12 receptor expression and IL-12 responsiveness J. Immunol. 161,2723-2730[Abstract/Free Full Text]
    25. 25
  25. Harris, S. G., Padilla, J., Koumas, L., Ray, D., Phipps, R. P. (2002) Prostaglandins as modulators of immunity Trends Immunol. 23,144-150[CrossRef][Medline]
    26. 26
  26. Visser, J., van Boxel-Dezaire, A., Methorst, D., Brunt, T., de Kloet, E. R., Nagelkerken, L. (1998) Differential regulation of interleukin-10 (IL-10) and IL-12 by glucocorticoids in vitro Blood 91,4255-4264[Abstract/Free Full Text]
    27. 27
  27. Miyaura, H., Iwata, M. (2002) Direct and indirect inhibition of Th1 development by progesterone and glucocorticoids J. Immunol. 168,1087-1094[Abstract/Free Full Text]
    28. 28
  28. Newton, C., McHugh, S., Widen, R., Nakachi, N., Klein, T., Friedman, H. (2000) Induction of interleukin-4 (IL-4) by Legionella pneumophila infection in BALB/c mice and regulation of tumor necrosis factor {alpha}, IL-6, and IL-1ß Infect. Immun. 68,5234-5240[Abstract/Free Full Text]
    29. 29
  29. Flesch, I. E., Wandersee, A., Kaufmann, S. H. (1997) IL-4 secretion by CD4+ NK1+ T cells induces monocyte chemoattractant protein-1 in early listeriosis J. Immunol. 159,7-10[Abstract]
    30. 30
  30. Hunter, S. A., Audette, C. A., Burstein, S. (1991) Elevation of brain prostaglandin E2 levels in rodents by {delta} 1-tetrahydrocannabinol Prostaglandins Leukot. Essent. Fatty Acids 43,185-190[CrossRef][Medline]
    31. 31
  31. Weidenfeld, J., Feldman, S., Mechoulam, R. (1994) Effect of brain constituent anandamide, a cannabinoid receptor agonist, on the hypothalamo-pituitary-adrenal axis in the rat Neuroendocrinology 59,110-112[Medline]
    32. 32
  32. Puder, M., Weidenfeld, J., Chowers, I., Nir, I., Conforti, N., Siegel, R. (1982) Corticotrophin and corticosterone secretion following {Delta}1-tetrahydrocannabinol, in intact and in hypothalamic deafferentated male rats Exp. Brain Res. 46,85-88[Medline]
    33. 33
  33. Marsicano, G., Lutz, B. (1999) Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain Eur. J. Neurosci. 11,4213-4225[CrossRef][Medline]
    34. 34
  34. Wenger, T., Ledent, C., Csernus, V., Gerendai, I. (2001) The central cannabinoid receptor inactivation suppresses endocrine reproductive functions Biochem. Biophys. Res. Commun. 284,363-368[CrossRef][Medline]
    35. 35
  35. Chiappelli, F., Manfrini, E., Franceschi, C., Cossarizza, A., Black, K. L. (1994) Steroid regulation of cytokines. Relevance for TH1-to-TH2 shift? Ann. N. Y. Acad. Sci. 746,204-215[Medline]
    36. 36
  36. Kalinski, P., Schuitemaker, J. H., Hilkens, C. M., Kapsenberg, M. L. (1998) Prostaglandin E2 induces the final maturation of IL-12-deficient CD1a+CD83+ dendritic cells: the levels of IL-12 are determined during the final dendritic cell maturation and are resistant to further modulation J. Immunol. 161,2804-2809[Abstract/Free Full Text]
    37. 37
  37. Cadepond, F., Ulmann, A., Baulieu, E. E. (1997) RU486 (mifepristone): mechanisms of action and clinical uses Annu. Rev. Med. 48,129-156[CrossRef][Medline]
    38. 38
  38. Valiveti, S., Hammell, D. C., Earles, D. C., Stinchcomb, A. L. (2004) In vitro/in vivo correlation studies for transdermal {delta}8-THC development J. Pharm. Sci. 93,1154-1164[CrossRef][Medline]
    39. 39
  39. Naef, M., Russmann, S., Petersen-Felix, S., Brenneisen, R. (2004) Development and pharmacokinetic characterization of pulmonal and intravenous {delta}-9-tetrahydrocannabinol (THC) in humans J. Pharm. Sci. 93,1176-1184[CrossRef][Medline]
    40. 40
  40. Zhang, D., Kishihara, K., Wang, B., Mizobe, K., Kubo, C., Nomoto, K. (1998) Restraint stress-induced immunosuppression by inhibiting leukocyte migration and Th1 cytokine expression during the intraperitoneal infection of Listeria monocytogenes J. Neuroimmunol. 92,139-151[CrossRef][Medline]
    41. 41
  41. Flint, M. S., Tinkle, S. S. (2001) C57BL/6 mice are resistant to acute restraint modulation of cutaneous hypersensitivity Toxicol. Sci. 62,250-256[Abstract/Free Full Text]
    42. 42
  42. Manzanares, J., Corchero, J., Fuentes, J. A. (1999) Opioid and cannabinoid receptor-mediated regulation of the increase in adrenocorticotropin hormone and corticosterone plasma concentrations induced by central administration of {delta}(9)-tetrahydrocannabinol in rats Brain Res. 839,173-179[CrossRef][Medline]
    43. 43
  43. Franchimont, D., Galon, J., Gadina, M., Visconti, R., Zhou, Y., Aringer, M., Frucht, D. M., Chrousos, G. P., O’Shea, J. J. (2000) Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes J. Immunol. 164,1768-1774[Abstract/Free Full Text]
    44. 44
  44. Mansilla-Rosello, A., Ferron-Orihuela, J. A., Ruiz-Cabello, F., Garrote-Lara, D., Fernandez-Mondejar, E., Delgado-Carrasco, M. L. (1997) Differential effects of IL-1 ß and ibuprofen after endotoxic challenge in mice J. Surg. Res. 67,199-204[CrossRef][Medline]
    45. 45
  45. Wenger, T., Moldrich, G. (2002) The role of endocannabinoids in the hypothalamic regulation of visceral function Prostaglandins Leukot. Essent. Fatty Acids 66,301-307[CrossRef][Medline]
    46. 46
  46. Wenger, T., Fernandez-Ruiz, J. J., Ramos, J. A. (1999) Immunocytochemical demonstration of CB1 cannabinoid receptors in the anterior lobe of the pituitary gland J. Neuroendocrinol. 11,873-878[CrossRef][Medline]
    47. 47
  47. Welch, S. P., Huffman, J. W., Lowe, J. (1998) Differential blockade of the antinociceptive effects of centrally administered cannabinoids by SR141716A J. Pharmacol. Exp. Ther. 286,1301-1308[Abstract/Free Full Text]
    48. 48
  48. Maccarrone, M., Bari, M., Battista, N., Finazzi-Agro, A. (2002) Endocannabinoid degradation, endotoxic shock and inflammation Curr. Drug Targets Inflamm. Allergy 1,53-63[CrossRef][Medline]
    49. 49
  49. Mirani, M., Elenkov, I., Volpi, S., Hiroi, N., Chrousos, G. P., Kino, T. (2002) HIV-1 protein Vpr suppresses IL-12 production from human monocytes by enhancing glucocorticoid action: potential implications of Vpr coactivator activity for the innate and cellular immunity deficits observed in HIV-1 infection J. Immunol. 169,6361-6368[Abstract/Free Full Text]
    50. 50
  50. Alesci, S., De Martino, M. U., Mirani, M., Benvenga, S., Trimarchi, F., Kino, T., Chrousos, G. P. (2003) L-carnitine: a nutritional modulator of glucocorticoid receptor functions FASEB J. 17,1553-1555[Abstract/Free Full Text]
    51. 51
  51. Betz, M., Fox, B. S. (1991) Prostaglandin E2 inhibits production of Th1 lymphokines but not of Th2 lymphokines J. Immunol. 146,108-113[Abstract]
    52. 52
  52. Kalinski, P., Hilkens, C. M., Snijders, A., Snijdewint, F. G., Kapsenberg, M. L. (1997) IL-12-deficient dendritic cells, generated in the presence of prostaglandin E2, promote type 2 cytokine production in maturing human naive T helper cells J. Immunol. 159,28-35[Abstract]
    53. 53
  53. Kuroda, E., Sugiura, T., Zeki, K., Yoshida, Y., Yamashita, U. (2000) Sensitivity difference to the suppressive effect of prostaglandin E among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice J. Immunol. 164,2386-2395[Abstract/Free Full Text]
    54. 54
  54. Li, J., Padigel, U. M., Scott, P., Farrell, J. P. (2002) Combined treatment with interleukin-12 and indomethacin promotes increased resistance in BALB/c mice with established Leishmania major infections Infect. Immun. 70,5715-5720[Abstract/Free Full Text]
    55. 55
  55. Hunter, S. A., Burstein, S. H. (1997) Receptor mediation in cannabinoid-stimulated arachidonic acid mobilization and anandamide synthesis Life Sci. 60,1563-1573[CrossRef][Medline]
    56. 56
  56. Klein, T. W., Newton, C., Larsen, K., Chou, J., Perkins, I., Lu, L., Nong, L., Friedman, H. (2004) Cannabinoid receptors and T helper biasing J. Neuroimmunol. 147,91-94[CrossRef][Medline]



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