Granule Formation in NGF-Cultured Mast Cells Is Associated with Expressions of Pyruvate Kinase Type M2 and Annexin I Proteins

Abstract

Background: Nerve growth factor (NGF) is a potent mediator, which regulates characteristics of mast cells, but its biological function is not well characterized. This study aimed to screen proteins associated with the maturation of human mast cells-1 (HMC-1) or mouse bone marrow-derived mast cells (BMMCs) cultured with NGF, and to examine the functions of proteins involved. Methods: NGF (10 ng/ml) was added to cell culture medium every other day for 10 days for HMC-1 or twice a week for 5 weeks for BMMCs. Granule formation was determined by electron microscopy or May-Grünwald-Giemsa staining, TNF-α by ELISA, expressions of various proteins by two-dimensional gel electrophoresis (2-DE), siRNA transfection by Lipofectamine 2000, and the expressions of pyruvate kinase and annexin I by immunoblotting. Results: After NGF treatment, granule formation and total amounts of granular mediator, TNF-α increased in both mast cells. This TNF-α was released by calcium ionophore or by antigen/antibody reaction. Expressions of pyruvate kinase and annexin I obtained by 2-DE were confirmed by immunoblotting and siRNA-transfected HMC-1 cells. Expressions of proteins, granule formation and TNF-α content were blocked by both the TrkA inhibitor, K252a, and the ERK inhibitor, PD98059, but not by the PI3 kinase inhibitors, LY294002 and wortmannin. Conclusion: These data suggest that pyruvate kinase and annexin I expressed by NGF contribute to granule formation containing TNF-α as well as other mediators in mast cells, which play a major role in allergic diseases via a TrkA/ERK pathway.

Cigarette smoke exacerbates mouse allergic asthma through Smad proteins expressed in mast cells

“…Numbers of infiltrated inflammatory cells or mast cells stained with Diff-Quick and May Grünwald-Giemsa [32], respectively, were quantified under microscopy (see additional file for details).…”

“…After BAL had been performed, lungs tissue sections embedded in paraffin (3 μm) were stained with hematoxylin and eosin (H & E) for general morphology, with periodic acid-Schiff (PAS) for identification of goblet cells [31], with van Gieson for collagen deposition [33] and with May Grünwald-Giemsa for mast cells [32] (see additional file for details). Collagen amounts were assessed by the Sircol collagen assay according to the manufacturer's instruction (see additional file for details) [34].…”

“…Bone marrow cells flushed from femurs and tibias of BALB/c mice (female, 8 wk old) were cultured in RPMI-1640 and 50% WEHI-3B conditioned media for 5 wk and confirmed as described previously [32] (see additional file for details).…”

“…The cells were washed and then challenged with 1.0 ng/ml DNP-HSA for time periods indicated (optimal time, 6 h) at 37°C in Tyrode's buffer [32]. CSE solution was treated for 6 h in DNP-HSA stimulation.…”

“…After incubation, BMMCs were added to the mixer. Transfected-BMMCs were activated with anti-DNP IgE antibody (0.1 μg/ml) and DNP-HSA (1.0 ng/ml) as methods described previously [32], and then the activities of MAP kinases, NF-κB, PAI-1 and expressions of cytokines were measured using western blot and RT-PCR, respectively.…”