Regulation of Antimicrobial Peptide and Ciprofloxacin Induced Mast Cell Responses by β-arrestin2

Objectives: Previous studies have shown that there is an increase in the number of mast cells that infiltrate gingival tissue in periodontal disease, mast cells contribute to porphyromonas gingivalis-induced bone loss in vivo, and human mast cells express the receptor MRGPRX2. These findings suggest that recruitment and activation of mast cells via MRGPRX2 could contribute to the pathogenesis of periodontal disease. MrgprB2 is the only mouse Mrg-GPCR that is expressed in mast cells. β-arrestin2 is an adapter molecule that serves as a negative regulator for GPCR-mediated neutrophil chemotaxis. The objective of my research is to determine if LL-37 activates murine mast cells via MrgprB2 and if β-arrestin2 regulates LL-37-induced mast cell responses.
Methods: Culture of Peritoneal-derived Mast Cells: Cell suspension was isolated from the peritoneal cavity of mice and cultured in growth medium and recombinant stem cell factor. After 2 weeks, toluidine blue staining was performed to confirm that >95% of the cells are mast cells.
PCR: To confirm the absence of the receptor in MrgprB2 ^-/^- and β-arrestin2 ^-/^- mast cells, PCR was used to determine the expression level of MrgprB2 and β-arrestin2.
Mast cell chemotaxis: Different concentrations of Ciprofloxacin and LL-37 (30 µl) or buffer were added to the lower wells of a 96-well chemotaxis chamber. Wild-type, MrgprB2 ^-/^- or β-arrestin2 ^-/^- mast cells were placed on the upper chamber. After 3 hour incubation at 37°C migrated cells were collected from the lower chambers. The cells were counted with a hemocytometer and expressed as absolute number of cells that had migrated.
Mast cell degranulation: Cells were stimulated with different concentrations of LL-37 and 48/80 and percent degranulation (β-hexosaminidase release) was measured using p-nitrophenyl-N-acetyl-b-D-glucosamine as substrate (absorbance at 405 nm).
Results: β-arrestin2 -/- showed less chemotaxis in response to Ciprofloxacin when compared to the wildtype. Murine peritoneal mast cells were stimulated with 48/80 and LL-37 and percent degranulation was determined. B-arrestin2 -/- cells showed increased degranulation compared to the wildtype upon stimulation. LL-37 induced more degranulation than 48/80.
Conclusions: β-arrestin2 serves as a negative regulator of mast cell degranulation and is required for mast cell chemotaxis.