Quantitative method to determine mRNA levels by reverse transcriptase-polymerase chain reaction from leukocyte subsets purified by fluorescence-activated cell sorting: Application to peripheral cannabinoid receptors

Marchand, Jean; Bord, A.; Pénarier, Géraldine; Lauré, F; Carayon, Pierre; Casellas, Pierre

doi:10.1002/(sici)1097-0320(19990301)35:3<227::aid-cyto5>3.0.co;2-4

Cited by 12 publications

(3 citation statements)

References 25 publications

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“…These results have been reproduced by Carayon et al [14] using flow-cytometry techniques to detect cell-surface receptors and by Marchand et al [15] using quantitative RT-PCR to detect CB2 transcripts. Immunohistological analysis of CB2 receptors in the tonsil showed that only B cells, not T cells, stained in the mantle of follicles.…”

Section: Cb2 Receptorsupporting

confidence: 54%

Cannabinoids, Immune System and Cytokine Network

Massi

Vaccani²,

Parolaro

2006

CPD

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How cannabinoids influence immune function has been examined extensively in the last 30 years. Studies on drug-abusing humans and animals, as well as in vitro models employing immune cell cultures, have shown that marijuana, natural and endogenous cannabinoid compounds are immunomodulators. These substances modulate host resistance to bacterial, protozoan and viral infections as well as they can profoundly affect the Th1/Th2 response. Recently, two types of cannabinoid receptor, CB1 and CB2, have been discovered. While CB1 is expressed primarily in the brain, CB2 is peculiar of the immune cells. Cannabinoid receptors have been shown to be involved in some but not all of immune effects. Nevertheless, their identification provides a specific mechanism of action in the attempting to find out how exogenous cannabinoids and endogenous cannabinoid system affect the immune apparatus, strengthen the hypothesis of cannabinoids as immunomodulators. As support to this theory, enough evidence exists to suggest that the cannabinoid system significantly affects almost every component of the immune response machinery and impacts the functioning also of the cytokine network. The evaluation of the biological consequences of these drug-induced cytokine changes has also dramatically become important considering not only the impact of cytokines on immune system per se but also envisaging their influence in cancer, inflammation, autoimmune disease, brain injury, hematopoietic colony formation in which cannabinoids have demonstrated a clear role as important modulators.

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Section: Cb2 Receptorsupporting

confidence: 54%

Cannabinoids, Immune System and Cytokine Network

Massi

Vaccani²,

Parolaro

2006

CPD

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“…The corresponding cDNA were amplified by polymerase chain reaction, and the products were quantified using a microplate hybridization assay as already described in detail (26).…”

Section: Methodsmentioning

confidence: 99%

Genomic and Functional Changes Induced by the Activation of the Peripheral Cannabinoid Receptor CB2 in the Promyelocytic Cells HL-60

Derocq

Jbilo

Bouaboula

et al. 2000

Journal of Biological Chemistry

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The function of the peripheral cannabinoid receptor (CB2), which is mainly expressed on hematopoietic cells, remains an enigma. In an attempt to decipher its role, we used Affymetrix TM Two G-protein-coupled receptors with seven transmembrane domains have been identified as cannabinoid receptors and are referred to as CB1 and CB2 (1-4). The central receptor CB1 is predominantly expressed in the brain, whereas the peripheral receptor CB2 is mainly expressed on cells of immune origin (5-7). In addition to psychotropic effects mediated by the central receptor, many reports have described a cannabinoid-induced modulation of immune functions, which could be attributed to the peripheral receptor subtype. With the exception of few examples reporting stimulating effects (8 -10), most of the studies showed immunosupressive properties of cannabinoids in very different areas of the immunity, including humoral and cellular responses as well as cytokine production (11-13). However many of these studies were conducted in the micromolar range higher than that required for receptor binding and often without controlling the specificity of the observed effects. As high concentrations of cannabinoids may induce nonspecific membrane perturbations (14), the question of the receptor specificity of the widespread suppressive effects reported so far is raised. Therefore, despite the characterization of the CB2 receptors on the immune cells (6), the identification of second messenger systems (15, 16), the finding of putative endogenous ligands (17)(18)(19), and the availability of specific antagonist ligands (20, 21), the function of CB2 receptors in the immune system is still elusive.In the present paper, to better define the complex involvement of this receptor in the immune functions, we used the recently developed DNA microarray technology to study the gene expression profile induced through the specific activation of the CB2 receptor. In a preceding study (22), we show that a stimulation of the CB2 receptor in HL-60 cells was able to induce the expression of the two chemokines IL-8 1 and MCP-1. In this study, using DNA microarrays allowing the differential evaluation of thousands of genes at the same time, we went a step further by providing a more complete gene expression profile specifically induced via the CB2 receptors in HL-60 cells. As a source of hematopoietic cells and myelomonocytic committed progenitors, these cells constitute a relevant model for the analysis of cell differentiation and the study of development of the immune effector functions. Moreover these cells offer the advantage of expressing the CB2 receptors only, excluding CB1 interferences. To compensate for the low density of CB2 receptors and to amplify the cannabinoid-induced effects, the receptor was overexpressed by transfection.Using this approach, we demonstrated for the first time that nanomolar concentrations of the cannabinoid ligand CP 55,940 induced the expression of factors mainly involved in gene transcription and cell differentiation processes ...

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“…These results were further confirmed at the mRNA level. TNF-␣ and INF-␥ gene expressions were determined by quantitative reverse transcription-PCR (22,23) in the early phase of the disease (day 10) in leukocytes extracted from the spinal cord of EAE-induced rats. As expected (19), TNF-␣ and IFN-␥ expressions were highly detected in leukocytes from rats with EAE, contrary to normal rats with barely detectable expression of the two cytokines (Fig.…”

Section: Sr 57746a Lowered Inflammatory Infiltration Into the Cnsmentioning

confidence: 99%