Nimita H. Fifadara

Chemokines play important regulatory roles in immunity, but their contributions to mast cell function remain poorly understood. We examined the effects of FcεRI–chemokine receptor (CCR) 1 co-stimulation on receptor localization and cellular morphology of bone marrow-derived mast cells. Whereas FcεRI and CCR1 co-localized at the plasma membrane in unsensitized cells, sensitization with IgE promoted internalization of CCR1 molecules. Co-stimulation of FcεRI and CCR1 with antigen and macrophage inflammatory protein-1α was more effective than FcεRI stimulation alone in causing leading edge formation, flattened morphology, membrane ruffles and ganglioside (GM1⁺) lipid mediator release. Co-stimulation resulted in phalloidin-positive cytoneme-like cellular extensions, also known as tunneling nanotubes, which originated at points of calcium accumulation. This is the first report of cytoneme formation by mast cells. To determine the importance of lipid rafts for mast cell function, the cells were cholesterol depleted. Cholesterol depletion enhanced degranulation in resting, sensitized and co-stimulated cells, but not in FcεRI-cross-linked cells, and inhibited formation of filamentous actin⁺ cytonemes but not GM1⁺ cytonemes. Treatment with latrunculin A to sequester globular-actin abolished cytoneme formation. The cytonemes may participate in intercellular communication during allergic and inflammatory responses, and their presence in the co-stimulated mast cells suggests new roles for CCRs in immunopathology.

Chemokine receptors (CCRs) are important co-stimulatory molecules found on many blood cells and associated with various diseases. The expression and function of CCRs on mast cells has been quite controversial. In this study, we report for the first time that murine bone marrow-derived mast cells (BMMC) express messenger RNA and protein for CCR1. BMMC cultured in the presence of murine recombinant stem cell factor and murine IL-3 expressed CCR1 after 5–6 weeks. We also report for the first time that mBMMC^CCR1+ cells endogenously express neurokinin receptor-1 and intercellular adhesion molecule-1. To examine the activity of CCR1 on these BMMC, we simultaneously stimulated two receptors: CCR1 by its ligand macrophage inflammatory protein-1α and the IgE receptor FcεRI by antigen cross-linking. We found that co-stimulation enhanced BMMC degranulation compared with FcεRI stimulation alone, as assessed by β-hexosaminidase activity (85 versus 54%, P < 0.0001) and Ca²⁺ influx (223 versus 183 nM, P < 0.05). We also observed significant increases in mast cell secretion of key growth factors, cytokines and chemokine mediators upon CCR1–FcεRI co-stimulation. These factors include transforming growth factor β-1, tumor necrosis factor-α and the cytokine IL-6. Taken together, our data indicate that CCR1 plays a key role in BMMC function. These findings contribute to our understanding of mechanisms for immune cell trafficking during inflammation.