Differential expression of the CB2 cannabinoid receptor by rodent macrophages and macrophage-like cells in relation to cell activation

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Abstract

An in vitro model of multi-step activation, in which cells of macrophage lineage are driven sequentially through inflammatory, primed, and fully activated states, was employed to assess for cannabinoid receptor expression. Murine and rat peritoneal macrophages, murine RAW264.7 and P388D1 macrophage-like cells, and neonatal rat brain cortex microglia expressed the cannabinoid receptor type 2 (CB2) differentially in relation to cell activation. The CB2 was undetectable in resident peritoneal macrophages, present at high levels in thioglycolate-elicited inflammatory and interferon gamma (IFNγ)-primed peritoneal macrophages, and detected at significantly diminished levels in bacterial lipopolysaccharide (LPS)-activated peritoneal macrophages. A comparable pattern of differential expression of the CB2 was noted for murine macrophage-like cells and neonatal rat brain cortex microglia. The cannabinoid receptor type 1 (CB1) was not detected in peritoneal macrophages or murine macrophage-like cells regardless of cell activation state but was present in neonatal rat microglia at low levels. These results indicate that levels of the CB2 in cells of macrophage lineage undergo major modulatory changes in relation to cell activation. Furthermore, since inflammatory and primed macrophages express the highest levels of CB2, the functional activities of macrophages when in these respective states of activation may be the most sensitive to the action of cannabinoids.

Introduction

Cannabinoids have been reported to modulate the functions of immune cells, including those of B and T lymphocytes, macrophages, and Natural Killer cells [1], [2]. Some of these effects have been implicated as linked functionally to cannabinoid receptors [3], [4]. To date, two cannabinoid receptors have been identified. The first, the cannabinoid receptor type 1 (CB1), is expressed at high levels in neural tissue [5] and testis [6] but can be found also at low levels in cells of the immune system [7]. The second, the cannabinoid receptor type 2 (CB2), has been identified primarily in cells of the immune system [7], [8] although recent reports indicate its presence also in adult uterine tissue and embryonic organs [9] and adult rat retina [10]. Both receptors are members of the large seven transmembrane G-protein-coupled receptor family and signal by inhibiting the production of the second messenger cAMP [11], [12], [13]. The CB2 is the cannabinoid receptor thought to be associated with modulation of immune functions [4], [14]. However, recent reports indicate that the CB1 also may be linked to cannabinoid-mediated alterations of immune cell activities [3], [15].

Macrophages serve as targets of cannabinoids. Functional activities of alveolar macrophages [16], as well as those of peritoneal macrophages [17], [18], have been shown to be altered by cannabinoids. In addition, cannabinoids have been reported to alter functional activities of microglia [3], [19], [20], cells which constitute a resident population of macrophages in the brain. These cells, typically of macrophage lineage, are capable of migration, differentiation, and proliferation [21]. In the adult, microglia are relatively quiescent and ramified in appearance [22]. However, during early development and after brain injury, they become activated and ameboid, phagocytose tissue debris [23] and produce cytokines such as interleukin 1β (IL-1β) [24] and interleukin 6 (IL-6) [25]. In addition, microglia have been shown to produce tumor necrosis factor α (TNFα) in culture [26]. Thus, microglia are capable of responding to extracellular stimuli in a mode which includes changes in morphology, differential gene expression, and exertion of distinctive functional activities similar to macrophages in the periphery at non-neuronal sites.

In the present study, we have used an in vitro model of macrophage multi-step activation [27], [28] to assess whether cannabinoid receptors are expressed differentially in relation to cell activation state. Resident peritoneal macrophages served as a source of “quiescent” or resting macrophages while thioglycolate-elicited macrophages served as a source of inflammatory macrophages. Resident and inflammatory macrophages were driven to a primed state with species-specific interferon gamma (IFNγ). Primed macrophages, in turn, were triggered to full activation with bacterial lipopolysaccharide (LPS). A similar process of exposure to IFNγ or LPS was used to drive RAW264.7 murine macrophage-like cells and purified neonatal rat brain cortex microglia to primed or fully activated states, respectively.

Assessment of gene expression for cannabinoid receptors was achieved by Mutational Reverse Transcription-Polymerase Chain Reaction (MRT-PCR), Ribonuclease Protection Assay (RPA), and Western immunoblotting. Our results indicate that the CB2 is expressed differentially in macrophages and macrophage-like cells in relation to cell activation state. High levels of CB2 were expressed by inflammatory and primed macrophages and macrophage-like cells, whereas minimal levels were expressed by these cells in their resident and fully activated states. This pattern of differential expression was observed for murine and rat peritoneal macrophages, murine macrophage-like cells, and neonatal rat brain cortex microglia maintained in vitro. These results suggest that differential expression of the CB2 is applicable to cells of macrophage lineage and that the nature of the macrophage functional response to cannabinoids is modulated by the activation state of the cell.

Section snippets

Reagents

All reagents, unless otherwise noted, were obtained from Sigma (St. Louis, MO). Restriction enzymes were purchased from New England BioLaboratories (Beverly, MA).

Cells, cell culture, and tissues

The murine macrophage-like cell lines RAW264.7 and P388D1 were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in complete RPMI 1640 medium (RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS)), 1% l-glutamine, nonessential amino acids, 100 μg/ml streptomycin, 100 U/ml

In vitro model of macrophage activation

Thioglycolate-elicited murine and rat peritoneal macrophages were maintained in medium, treated with IFNγ, or treated with LPS in the presence or absence of IFNγ to establish, respectively, in vitro correlates of responsive inflammatory, primed, or “fully” activated states. Resident peritoneal macrophages served as a source of non-inflammatory macrophages. Similarly, murine macrophage-like cells RAW264.7 and P388D1 and neonatal rat brain cortex microglia were treated with IFNγ or with LPS in

Discussion

There have been numerous reports that cannabinoids alter the functional activities of immune cells [1], [2]. Recent studies suggest that some of these effects are mediated by cannabinoid receptors [4], [15]. To date, two cannabinoid receptors have been identified: the CB1, which is distributed primarily in brain and nerve tissue [5], [32], [33], and the CB2, which is expressed primarily in cells of the immune system [7], [8]. While a large body of data is available concerning the pharmacology

Acknowledgements

The authors thank Ms. C. Boothe for technical expertise in the preparation of macrophage and microglial cultures and Dr. K. Nowell Harmon for scanning electron microscopy. This research was supported by NIH awards: DA05832, DA05274, and T32DA07027.

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