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. 2013 Jun;21(3):253-9.
doi: 10.1007/s10787-012-0133-9. Epub 2012 Apr 11.

Differential Migratory Properties of Monocytes Isolated From Human Subjects Naïve and Non-Naïve to Cannabis

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Differential Migratory Properties of Monocytes Isolated From Human Subjects Naïve and Non-Naïve to Cannabis

Michelle Sexton et al. Inflammopharmacology. .
Free PMC article

Abstract

This study evaluates the migratory potential of monocytes isolated from two groups of human subjects: naïve and non-naïve to Cannabis. Phytocannabinoids (pCB), the bioactive agents produced by the plant Cannabis, regulate the phenotype and function of immune cells by interacting with CB1 and CB2 receptors. It has been shown that agents influencing the phenotype of circulating monocytes influence the phenotype of macrophages and the outcome of immune responses. To date, nothing is known about the acute and long-term effects of pCB on human circulating monocytes. Healthy subjects were recruited for a single blood draw. Monocytes were isolated, fluorescently labeled and their migration quantified using a validated assay that employs near infrared fluorescence and modified Boyden chambers. CB1 and CB2 receptor mRNA expression was quantified by qPCR. Monocytes from all subjects (n = 10) responded to chemokine (c-c motif) ligand 2 (CCL2) and human serum stimuli. Acute application of pCB significantly inhibited both the basal and CCL2-stimulated migration of monocytes, but only in subjects non-naïve to Cannabis. qPCR analysis indicates that monocytes from subjects non-naïve to Cannabis express significantly more CB1 mRNA. The phenotype of monocytes isolated from subjects non-naïve to Cannabis is significantly different from monocytes isolated from subjects naïve to Cannabis. Only monocytes from subjects non-naïve to Cannabis respond to acute exposure to pCB by reducing their overall migratory capacity. Our study suggests that chronic exposure to Cannabis affects the phenotype of circulating monocytes and accordingly could influence outcome of inflammatory responses occurring in injured tissues.

Conflict of interest statement

Conflict of interest The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
CCL2 increases the migration of freshly isolated human monocytes. a Correlation between cell quantity and fluorescence units: freshly isolated monocytes were labelled with DRAQ5 and phalloydin. After incubation and migration, non-migrated cells were wiped from the top side of the filter, rinsed with PBS and fluorescence values for migrated cells (bottom of the filter) were quantified on the Odyssey® imaging system. After fluorescence scanning, membranes were manually counted on a Zeiss Axiovert microscope. Average number of cells in three representative fields per condition correlated well with fluorescence values. b CCL2 is necessary for serum-induced migration. Using the potent chemo-attractant, CCL2, and human serum, we established positive controls with CCL2 and serum. Applying a CCL2 neutralizing antibody to the conditioned media in the lower chambers inhibited this migratory induction to basal levels. As a control, neutralizing antibody on its own showed no inhibitory activity on migration
Fig. 2
Fig. 2
Differential migratory responses of monocytes isolated from Cannabis naïve and non-naïve subjects. Monocytes isolated from five subjects (n = 3 for each condition), who were naïve to Cannabis use, have a significantly increased response to CCL2 and serum compared to monocytes isolated from subjects who were non-naïve to Cannabis use. Significance p < 0.05 (students’ t test,)
Fig. 3
Fig. 3
Differential sensitivity to pCB between Cannabis naïve and non-naïve subjects. (a) Basal migration of freshly isolated human monocytes was significantly reduced by addition of phytocannabinoids (pCB) to the lower wells of the migration chamber: THC, cannabidiol (CBD), cannabinol (CBN) and a set ratio of these compounds (MIX). Basal migration was significantly inhibited in the non-naïve cohort. (b) Additionally, the migratory response toward CCL2 was also reduced by ~25 % when the pCBs were added to CCL2 in the lower chambers. In control experiments (not shown) pCBs alone in the upper chamber with cell loading did not affect basal migration. Statistical significance ***p < 0.001; **p < 0.001; *p < 0.05 (unpaired, two-tailed t test calculated by GraphPad)
Fig. 4
Fig. 4
Cannabinoid receptor message in human monocytes: a subjects non-naïve to Cannabis have fourfold increase in CB1 receptor mRNA compared to naïve subjects. Total RNA was amplified using qPCR and ΔΔCT calculations using TBS as a control housekeeping gene was used for quantification. Human brain RNA was used as a positive control and calibration sample. The difference did not reach statistical significance. b No difference was found in mRNA for CB2 receptor between the cohorts. Human spleen RNA was used as a positive control (RQ relative quantification)

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