Elsevier

Biochemical Pharmacology

Volume 82, Issue 4, 15 August 2011, Pages 380-388
Biochemical Pharmacology

Endocannabinoids inhibit release of nerve growth factor by inflammation-activated mast cells

https://doi.org/10.1016/j.bcp.2011.05.004Get rights and content

Abstract

Nerve growth factor (NGF) is a pleiotropic member of the neurotrophin family. Beside its neuronal effects, NGF plays a role in various processes, including angiogenesis. Mast cells release NGF and are among elements contributing to angiogenesis, a process regulated by arrays of factors, including the inhibitory cannabinoids. The possible inhibitory role of cannabinoids on mast cell-related NGF mitogenic effect on endothelial cells was then investigated. Human mastocytic cells HMC-1, challenged with PMA to yield release of NGF, were preincubated with the endocannabinoid PEA. Then, conditioned media were added to HUVEC cultures. PMA-activated HMC-1 cells released substantial amounts of NGF, whereas PEA inhibited PMA-induced NGF release. HUVEC proliferation increased after treatment with media from activated HMC-1 cells, while was reduced with media from HMC-1 cells treated with PEA. To characterize receptors mediating such effects of PEA, RT-PCR and western blot analysis were performed on HMC-1 cells. None of the two cannabinoid CB1 and CB2 receptors was expressed by HMC-1 cells, which on the other hand expressed the orphan receptor GPR55. PEA was ineffective in inhibiting NGF release from HMC-1 cells treated with PMA and transfected with positive GPR55 RNAi, whereas it induced significant reduction of NGF in cells transfected with the corresponding negative control RNAi. Results indicate that NGF released from inflammatory mast cells induces angiogenesis. Cannabinoids attenuate such pro-angiogenic effects of NGF. Finally, cannabinoids could be considered for antiangiogenic treatment in disorders characterized by prominent inflammation.

Introduction

Nerve growth factor (NGF) is the prototype molecule for the family of neurotrophins [1], of primary relevance to neurogenesis [2], neuronal repair processes [3], as well as neuronal plasticity [4] and survival.

Although neurotrophins are thought to exert their actions predominantly on neural cells [5], indeed some of them possess pleiotropic properties [6]. For example, NGF is able to exert pro-inflammatory effects [7] and displays prominent pro-angiogenic properties in vitro and in vivo [8].

Moreover, NGF can influence development and activation of an array of haematopoietic cell types, including basophiles and mast cells [9]. Interestingly, the latter can release substantial amounts of NGF stored in their granules [10]. Mast cells are derived from their haematopoietic precursors and represent critical effectors cells in allergic disorders and other IgE-dependent immune responses [11]. More recently, it has been shown that mast cells are also involved in the regulation of angiogenesis [12], the process leading to formation of new vessels starting from pre-existing ones [13].

From the biologic point of view, angiogenesis is the result of the concerted, balanced action of an array of stimulatory factors, such as, for instance, VEGF or FGF [14], or inhibitory factors of various nature, including angiostatin [15], endostatin [16], and the family of ubiquitary lipid mediators known as endocannabinoids [17], [18].

Endocannabinoids usually induce inhibitory responses in different tissues and organs [18], their effects being mediated by specific membrane receptors, such as those belonging to the CB1/CB2 system [19], [20], vanilloid receptors [21], and the TRPV1 receptor. Recently, a novel receptor, which is able to bind endocannabinoids, GPR55, has been identified and cloned [22].

In this paper, we describe the role of NGF released by noxious stimuli-activated mast cells in fuelling angiogenesis and, eventually, the modulating effects of the endocannabinoid PEA, an N-acylamidic molecule that negatively influences mast cell functions sustaining an inflammatory response [23].

To demonstrate the hypothesized angiogenic potential of mast cells, we first challenged the one-of-a kind human cell line, derived from a patient with mast cell leukaemia, mastocytic cells HMC-1 [24] with inflammatory stimuli and then measured NGF released in the media. Subsequently, human umbilical cord vascular endothelial cells (HUVEC) underwent challenge with conditioned media from activated mast cells.

Finally, in light of the described inhibitory effects of cannabinoids upon inflammation [18], we evaluated possible inhibitory effects of PEA on mast cells-related NGF release.

Section snippets

Cell cultures and reagents

HMC-1 cells (kindly provided by Dr. Butterfield, Rochester, MN), which are immature human leukemic mast cells, were cultured in Iscove's medium, supplemented with 10% fetal calf serum (FCS) (Invitrogen Srl, San Giuliano Milanese, Italy), 10 mM monothiolglycerol (Sigma–Aldrich, Milano, Italy), and antibiotics (streptomycin and penicillin) (Sigma–Aldrich, Milano, Italy). Stimulation of HMC-1 cells was performed in 24-multiwell plates (Falcon, Lincoln Park, NJ). Before stimulation, cells were

Nerve growth factor is substantially released by human mast cells challenged with inflammatory stimuli

In order to assess the ability of the human mast cell line HMC-1, cultures were incubated 24 h with graded concentrations of the pro-inflammatory compound phorbol myristate acetate (PMA), an activator of protein kinase C. PMA induced robust release of NGF by HMC-1 cells in a concentration-dependent manner. The most effective NGF releasing concentration of PMA was 50 ng/ml (Fig. 1, panel A). The release induced by PMA (50 ng/ml) was also time dependent, as incubation of HMC-1 cells resulted in

Discussion

In this paper, we demonstrate that nerve growth factor release from mast cells activated by inflammatory stimuli is inhibited in the presence of PEA, a lipid molecule belonging to the endocannabinoid family.

It is known that NGF is released by activated immunocytes and may mediate inflammatory reactions [27]. In this line, an increased number of polymorphonucleate cells are observed in animal models of inflammation [28], and the neurotrophin possesses hyperalgesic properties as assessed by

Acknowledgements

This work was accomplished thanks to generous contribution of the Epitech Group Inc., Abano Terme, and, in part, with the funds granted by MIUR to the PhD school in Preclinical and Clinical Pharmacology, University of Catania School of Medicine.

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