Impaired Mast Cell Maturation and Degranulation and Attenuated Allergic Responses in Ndrg1-Deficient Mice

Abstract

We have previously reported that N-myc downstream regulated gene-1 (NDRG1) is an early inducible protein during the maturation of mouse bone marrow-derived mast cells (BMMCs) toward a connective tissue mast cell-like phenotype. To clarify the function of NDRG1 in mast cells and allergic responses, we herein analyzed mast cell-associated phenotypes of mice lacking the Ndrg1 gene. Allergic responses including IgE-mediated passive systemic and cutaneous anaphylactic reactions were markedly attenuated in Ndrg1-deficient mice as compared with those in wild-type mice. In Ndrg1-deficient mice, dermal and peritoneal mast cells were decreased in number and morphologically abnormal with impaired degranulating ability. Ex vivo, Ndrg1-deficient BMMCs cocultured with Swiss 3T3 fibroblasts in the presence of stem cell factor, a condition that facilitates the maturation of BMMCs toward a CTMC-like phenotype, displayed less exocytosis than replicate wild-type cells after the cross-linking of FcεRI or stimulation with compound 48/80, even though the exocytotic response of IL-3-maintained, immature BMMCs from both genotypes was comparable. Unlike degranulation, the production of leukotriene and cytokines by cocultured BMMCs was unaffected by NDRG1 deficiency. Taken together, the altered phenotypes of Ndrg1-deficient mast cells both in vivo and ex vivo suggest that NDRG1 has roles in the terminal maturation and effector function (degranulation) of mast cells.

Analysis of Two Major Intracellular Phospholipases A2 (PLA2) in Mast Cells Reveals Crucial Contribution of Cytosolic PLA2α, Not Ca2+-independent PLA2β, to Lipid Mobilization in Proximal Mast Cells and Distal Fibroblasts

“…2F). Even after coculture with fibroblasts for 1 week, a period sufficient to induce functional maturation of mast cells (11)(12)(13)(14), IgE/Ag-stimulated release of ␤-HEX (Fig. 2G) (Fig.…”

“…Maturation of immature BMMCs toward CTMC-like cells was described previously in detail (11)(12)(13)(14). Briefly, 5 ϫ 10 6 BMMCs were seeded on a subconfluent Swiss 3T3 fibroblast monolayer in 100-mm culture dishes and cocultured for up to 4 days in the presence of the baculovirus/insect cell-produced soluble form of SCF with replacement of the medium every 2 days.…”

“…The PCR thermal conditions were as follows: 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s (for cPLA 2 s); 94°C for 30 s, 56°C for 30 s, and 72°C for 40 s (for iPLA 2 ␤), and 94°C for 30 s, 59°C for 30 s, and 72°C for 30 s (for mMCP-4). RT-PCR for mouse GAPDH was performed as described previously (14). After 28 cycles of amplification, the PCR products were run on 1% agarose gels with ethidium bromide.…”

“…One day later, the mice were intravenously challenged with 60 ng of Ag in 200 l of PBS containing 0.8% (w/v) Evans blue (Sigma). After 30 min, their ears were removed and dissolved, and the dye extravasation was determined colorimetrically at 630 nm, as described previously (14).…”

“…Culture and Activation of BMMCs, Macrophages, and Dendritic Cells-To prepare BMMCs, bone marrow cells were obtained from femurs and tibias of mice and cultured in IL-3-containing BMMC complete medium consisting of DMEM, 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 g/ml streptomycin, 100 mM nonessential amino acids, and an optimal amount of recombinant mouse IL-3 that had been produced by the baculovirus/insect cell system, as described previously (11)(12)(13)(14). After 4 -5 weeks of culture, the majority of floating cells was confirmed to be immature BMMCs as assessed by Alcian blue-positive and safranin O-negative staining of their granules.…”