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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 22, 2004 as doi:10.1096/fj.04-2190fje. |
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Center for Experimental Pathology, Istituto Cantonale di Patologia, Locarno, Switzerland
1Correspondence: Center for Experimental Pathology, Istituto Cantonale di Patologia, Via In Selva, 24 P.O. Box, Locarno 6601, Switzerland. E-mail: georges.maestroni{at}ti.ch
SPECIFIC AIMS
The decision-making mechanisms that determine the choice of the appropriate effector immune response to a microbial challenge are poorly understood. Microbial products stimulate toll-like receptors (TLRs) in DCs activating the innate immune response to produce inflammatory mediators and modulating expression of co-stimulatory molecules and the type and kinetic of cytokine produced. Nevertheless, the local microenvironment which may differ according to the route of entry of the infectious agent, may affect these mechanisms.
Endogenous cannabinoid such as anandamide and 2-AG may be produced in most tissues upon a variety of stimuli including TLRs agonist and traumatic injury. These substances act as physiological ligands for the G protein-coupled cannabinoid receptors CB1 and CB2, with CB2 receptors being expressed mostly in immunocompetent cells, including human DCs.
We studied the influence of 2-AG when injected during primary immunization on the recall memory immune response to a soluble protein.
PRINCIPAL FINDINGS
1. In vivo effect on Th priming
A soluble foreign protein was injected in combination with peptidoglycan (PGN) from S. Aureus to provide a danger signal to DCs. TLR2 recognizes the highest number of microbial products and PGN is a TLR2 agonist that has been reported to instruct myeloid DCs to induce a Th2 response. Mice were injected intradermally (id) with 100 µL of PBS containing key-hole limpet haemocyanin (50 µg, KLH) and PGN (1 µg) in the presence or absence of 2-AG (50 µM). Ten days later, the mice were injected subcutaneously with KLH (50 µg) in CFA. After seven days the mice were challenged with KLH and the delayed hypersensitivity response (DTH) and cytokine production in draining lymph nodes were assayed 48 h later. Figure 1
shows that the presence of 2-AG resulted in a significantly higher DTH response upon the recall memory response (Fig, 1A
) and in a strong inhibition of IL-4 production (Fig. 1B
). The production of IFN-
remained unchanged (Fig. 1B
). These effects were neutralized when the specific CB2 antagonist SR 144528 was injected together with 2-AG in the primary immunization. 2-AG exerted a Th1 skewing effect during primary immunization via CB2 receptors.
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2. Expression of CB2 receptor in murine DCs subsets
We investigated the CB2 receptor mRNA expression by real time reverse transcribed polymerase chain reaction in lymphoid CD8+, CD11c+ and CD8-, CD11c+ DCs populations. We found that CD8+CD11c+ DCs showed the highest expression of CB2 mRNA. CB2 mRNA expression was reduced to the brain level upon maturation by PGN. The reduced CB2 mRNA expression in mature DCS did not correlate with the CB2 protein expression that was similar in both immature and mature DCs.
3. 2-AG effect on DCs migration
As 2-AG was reported to act as chemoattractant for human macrophages and monocytes, we thought that the effect of 2-AG may be related to an enhanced migration of DCs from the site of antigen deposition to the draining lymph nodes. Therefore, we thought that the effect of 2-AG may be related to an enhanced migration of DCs and antigen presentation. To challenge this hypothesis we injected OVA-FITC (50µg) as foreign protein ± 2-AG and 24 h later we enumerated the CD11c+FITC+ and the CD8+FITC+ cells in the CD11c+-enriched cell population from the draining lymph nodes. 2-AG increased the number of DCs loaded with OVA-FITC in the draining lymph nodes, with the CD8+FITC+ subpopulation being more affected. When the CD11c+FITC+ general population was considered, the increase in migration was 163% (P<0.03). When considering the CD8+FITC+ subset only, the figure rose to 364% (P<0.01). The presence of SR 144528 counteracted completely these effects, indicating that the increase in the antigen-loaded DCs was a CB2-mediated phenomenon. 2-AG exerted a CB2-mediated powerful chemotactic activity on both immature and mature DCs, in vitro.
CONCLUSIONS AND SIGNIFICANCE
We suggest that the endocannabinoid 2-AG may act as chemotactic substance capable of recruiting DCs and/or their precursor cells during the innate immune response, which in the presence of a TLR2 agonist instruct a Th1 adaptive response. The apparent Th1 adjuvant effect of 2-AG seems to result from a reduced Th2 priming (decrease of IL-4 production) rather than from an increased Th1 polarization (no increase of IFN- production). As this effect would be consistent with both increased migration and type of DCs subset recruitment, we suggest that 2-AG preferentially recruits CD8+ DCs precursors and/or creates a microenvironment in vivo that accelerates DCs’ migration and instructs a Th1 response to a foreign soluble protein.
Cannabinoids have been reported to exert both anti-inflammatory and pro-inflammatory effects resulting in a complex and controversial picture. However, most in vivo studies related to systemic and direct effects on T and B cell responses of pharmacological treatments with the primary psychoactive component of marijuana, 
9-tetrahydrocannabinol and related substances. We believe to have revealed a potentially important role of 2-AG, which might have several implications. For example, 2-AG is induced in the central nervous system by traumatic brain injury at tissue concentrations largely above that used in our study. Its pathophysiological relevance seems to be that of attenuating brain damage via a CB1-mediated mechanism in a model of closed head injury where no exogenous danger signals were involved. Perhaps in the presence of a danger signal (infection) or inflammation, the effect of 2-AG in the brain would promote neuroinflammatory diseases. The ability of 2-AG to shape the innate immune response so that the ensuing adaptive response is Th1 skewed may find application in vaccine development.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2190fje;
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