Internalization of benzylisoquinoline alkaloids by resting and activated bone marrow-derived mast cells utilizes energy-dependent mechanisms

Syed Benazir Alam,Marianna Kulka

doi:10.1007/s00011-021-01526-2

Abstract

Objective and designDrug delivery to inflammatory cells is dependent upon poorly understood, complex endocytic processes. Berberine (BBR), a benzylisoquinoline alkaloid, binds to heparin and targets glycosaminoglycan-rich granules in mast cells (MC), but the mechanism of BBR internalization is unknown.MethodsBMMC were treated with various concentrations of BBR for different amounts of time and BBR internalization was assessed by flow cytometry and fluorescence microscopy. BMMC were pretreated with endocytic inhibitors or a growth factor (IL-3) prior to BBR exposure to access mechanisms of its internalization.ResultsAfter 24 h, 48 ± 0.8% of BMMC internalized BBR and this process was dependent upon temperature and the presence of glucose in the medium. Methanol fixation reduced BBR internalization, suggesting the involvement of an energy-dependent active transport mechanism. To determine mode of internalization, BBR was encapsulated into Lipofectamine TM lipoplexes since these are known to circumvent classical endocytic pathways. Incorporating BBR into lipoplexes decreased BBR internalization by 26% and 10% (10 μg/ml and 100 μg/ml Lipo-BBR respectively) by BMMC. BBR endocytosis was significantly reduced by Latrunculin B (88%), Cytochalasin B (87%), Chloroquine (86.5%) and 3-methyladenine (91%), indicating that actin polymerization, lysosomal pH and lysosomal self-degradation via the autophagy pathway was involved. In contrast, IL-3 treatment significantly enhanced BBR endocytosis (54% by 40 ng/ml IL-3) suggesting that IL-3 signaling pathways play a role in internalization.ConclusionsOur data suggests that internalization of BBR by resting and IL-3-activated BMMC utilizes an energy-dependent pathway that is dependent upon glucose metabolism and temperature. Furthermore, this process requires actin polymerization and lysosomal trafficking. These data suggest internalization of benzylisoquinoline compounds is an active and complex process.

Highlights

Mast cells are large granular immune cells that orchestrate homeostatic processes during inflammation, fibrosis, and infection [6]
To measure cell-associated BBR fluorescence, bone marrowderived mouse mast cells (BMMC) were washed twice with phosphate buffered saline (PBS)-bovine serum albumin (BSA) and resuspended in 70 ul Differentiated BMMC treated with berberine became fluorescent
Two of the most quintessential biomarkers of mast cells are the high affinity IgE receptor, FcεRI, which facilitates BMMC responses to antigens and the stem cell factor receptor, c-Kit (CD117), which is required for mast cell survival and differentiation

Summary

Introduction

Mast cells are large granular immune cells that orchestrate homeostatic processes during inflammation, fibrosis, and infection [6]. Kulka and immune responses to infection [7] Within their densely packed intracellular granules, mast cells store several immunomodulatory mediators including tryptase, histamine, cytokines and chemokines within a proteoglycan matrix. Receptor-mediated endocytosis of the high affinity mast cell IgE receptor, FcεRI, is certainly an important process for its activation [2] and is dependent upon membrane gangliosides such as GD1b [3]. The process of mast cell internalization of small molecules, inhibitors or therapeutic drugs, is even less clear. Since drug delivery applications rely heavily on these internalization processes, it is important to better understand mast cell internalization of such small molecules

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