Mast Cell Activation and Microtubule Organization Are Modulated by Miltefosine Through Protein Kinase C Inhibition.

Zuzana Rubíková,Pavel Dráber,Vadym Sulimenko,Tomáš Paulenda

doi:10.3389/fimmu.2018.01563

Abstract

Mast cells play an effector role in innate immunity, allergy, and inflammation. Antigen-mediated activation of mast cells initiates signaling events leading to Ca2+ response and the release of inflammatory and allergic mediators from granules. Diseases associated with deregulated mast cell functions are hard to treat and there is an increasing demand for new therapeutic strategies. Miltefosine (hexadecylphosphocholine) is a new candidate for treatment of mast cell-driven diseases as it inhibits activation of mast cells. It has been proposed that miltefosine acts as a lipid raft modulator through its interference with the structural organization of surface receptors in the cell membrane. However, molecular mechanisms of its action are not fully understood. Here, we report that in antigen-activated bone marrow-derived mast cells (BMMCs), miltefosine inhibits degranulation, reorganization of microtubules, as well as antigen-induced chemotaxis. While aggregation and tyrosine phosphorylation of IgE receptors were suppressed in activated cells pre-treated with miltefosine, overall tyrosine phosphorylation levels of Lyn and Syk kinases, and Ca2+ influx were not inhibited. In contrast, lipid raft disruptor methyl-β-cyclodextrin attenuated the Ca2+ influx. Tagged-miltefosine rapidly localized into the cell interior, and live-cell imaging of BMMCs with labeled intracellular granules disclosed that miltefosine inhibited movement of some granules. Immunoprecipitation and in vitro kinase assays revealed that miltefosine inhibited Ca2+- and diacylglycerol-regulated conventional protein kinase C (cPKC) isoforms that are important for mast cell degranulation. Inhibition of cPKCs by specific inhibitor Ly333531 affected activation of BMMCs in the same way as miltefosine. Collectively, our data suggest that miltefosine modulates mast cells both at the plasma membrane and in the cytosol by inhibition of cPKCs. This alters intracellular signaling pathway(s) directed to microtubules, degranulation, and migration.

Highlights

Mast cells play a pivotal role in innate immunity, allergy, and inflammation
To get deeper insight into the function(s) of miltefosine in mast cells we evaluated early stages of cell activation after cross linking of high affinity IgE receptor (FcεRI), Ca2+ influx, degranulation, microtubule reor ganization, and migration in bone marrow-derived mast cells (BMMCs) treated with miltefosine
To test whether the same holds true for mouse BMMCs, the degree of degranulation in miltefosine pre-treated and acti vated BMMCs was determined

Summary

Introduction

Diseases associated with deregulated mast cell fun ctions are hard to treat, and so the demand for new and better treatments targeting mast cell activation pathways increases. Mast cells express on their surfaces receptors with a high affinity for IgE (FcεRIs). Aggregation of FcεRIs by multivalent antigen (Ag)-IgE complexes leads to activation of signaling pathways resulting in the release of Ca2+ from the endoplasmic reticulum (ER) and subsequent activation of store-operated Ca2+ entry (SOCE). Mast cell activation by FcεRI aggregation is accompanied with changes in cell morphology, enhanced adhesion, and migration. It was reported that activation of mast cells induces increased formation of microtubules [2, 3] and their reorganization into protrusions containing microtubules (microtubule protrusions) [4, 5]. Independent of FcεRI aggregation, the activation events can be mimicked by non-specific activators, such as protein tyrosine phosphatase inhibitor pervanadate, inhibitor of ER Ca2+-ATPase pumps thapsigargin [4], or calcium ionophore A23187 [6]

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