Abstract
Background: IgE/allergen-activated murine mast cells (MCs) release potent granule-stored mediators by degranulation and secrete newly synthesized chemokines to locally recruit inflammatory cells. Whereas the fusion and trafficking steps during degranulation have been extensively studied, chemokine vesicular trafficking remains poorly understood.
Objective: We explored the trafficking of C-C motif chemokine ligand 2 (CCL2) in IgE-activated MCs and in a murine model of passive cutaneous anaphylaxis (PCA).
Methods: Bone marrow-derived and peritoneal cell-derived MCs from wild-type and newly established CCL2-enhanced green fluorescent protein knock-in mice were sensitized with anti-DNP (2,4-dinitrophenol) IgE and activated with DNP-human serum albumin antigen. Using time-course experiments, fluorescence-activated cell sorting, ELISA, and confocal and fluorescence microscopy analyses, the CCL2 vesicular trafficking in and secretion from MCs were analyzed. In vivo experiments were conducted to analyze CCL2 secretion during PCA.
Results: Cultured MCs released CCL2 in a biphasic manner, exocytosing vesicular prestored chemokine followed by secretion of neosynthesized protein that partly trafficked through vesicular-associated membrane protein (VAMP) 3- and VAMP7-positive vesicular carriers. CCL2 trafficking in soluble antigen-activated CCL2-enhanced green fluorescent protein knock-in MCs followed a complex pattern-polarized, multidirectional, or pseudopodia targeted-regulated by the interplay between the actin and microtubule cytoskeletons. Secreted CCL2 interacted with proteoglycans released during degranulation. On polarized activation by immobilized antigen, prestored CCL2 was secreted at the degranulatory interface, whereas neosynthesized CCL2 trafficked away from it. During PCA, CCL2 rapidly appeared lining blood vessels after MC degranulation and was locally released in the surrounding tissue.
Conclusion: Our study reveals the complex dynamics of chemokine secretion and trafficking driving local inflammation during MC-driven allergic responses.
Keywords: CCL2; chemokine; mast cells; passive cutaneous anaphylaxis; secretion; trafficking.