Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability

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Morin inhibits Fyn kinase in mast cells and IgE-mediated type I hypersensitivity response in vivo

Mast cells are responsible for IgE-mediated allergic responses. Although dietary flavonoid morin has been known to suppress mast cell activation, its in vivo anti-allergic activity and the underlying mechanisms remain are largely unknown. In this study, we determine whether morin suppresses IgE-mediated allergic responses in an animal model and its mechanism of action. Morin suppressed IgE-mediated PCA in mice (ED₅₀ 23.9 mg/kg) and inhibited degranulation and production of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-4 in antigen (Ag)-stimulated mast cells. The mechanism of action was a follows. Morin inhibited the activating phosphorylation of spleen tyrosine kinase (Syk) and linker for activation of T cells (LAT) in rat basophilic leukemia (RBL)-2H3 cells and bone marrow-derived mast cells (BMMCs). Akt and the mitogen-activated protein (MAP) kinases, p38, extracellular signal-regulated kinase (ERK)1/2, and c-Jun N-terminal kinase (JNK) were inhibited as well. In vitro kinase assay indicated that Fyn kinase, not Lyn and Syk, was inhibited by morin in a dose-dependent manner (IC₅₀ 5.7 μM). In conclusion, the results suggest that morin suppresses the IgE-mediated allergic response by primarily inhibiting Fyn kinase in mast cells.     

Identification of inducible genes during mast cell differentiation

Mast cells play an important role in allergic inflammation by releasing their bioactive mediators. The function of mast cells is enhanced by stimulation because of the induction of specific genes and their products. While many inducible genes have been elucidated, we speculated that a significant number of genes remain to be identified. Thus, we applied differential display (dd) PCR to establish a profile of the induced genes in bone marrow-derived mast cells (BMMCs) after they were co-cultured with 3T3 fibroblasts. To date, 150 cDNA fragments from the connective-type mast cells (CTMCs) were amplified. Among them, thirty cDNA fragments were reamplified for cloning and sequencing. The ddPCR strategy revealed that serine proteases were the most abundant genes among the sequenced clones induced during the maturation. Additionally, unknown genes from the co-culture of BMMCs with 3T3 fibroblasts were identified. We confirmed their induction in the CTMCs by Northern blot analysis and RT-PCR. Characterization of these induced genes during the maturation processes will provide insight into the functions of mast cells.     

Emodin, a naturally occurring anthraquinone derivative, suppresses IgE-mediated anaphylactic reaction and mast cell activation

The high-affinity receptor for IgE (Fc?RI)-mediated activation of mast cells plays an important role in allergic diseases such as asthma, allergic rhinitis and atopic dermatitis. Emodin, a naturally occurring anthraquinone derivative in oriental herbal medicines, has several beneficial pharmacologic effects, such as anti-cancer and anti-diabetic activities. However, the anti-allergic effect of emodin has not yet been investigated. To assess the anti-allergic activity of emodin, in vivo passive anaphylaxis animal model and in vitro mouse bone marrow-derived mast cells were used to investigate the mechanism of its action on mast cells. Our results showed that emodin inhibited degranulation, generation of eicosanoids (prostaglandin D₂ and leukotriene C₄), and secretion of cytokines (TNF-α and IL-6) in a dose-dependent manner in IgE/Ag-stimulated mast cells. Biochemical analysis of the Fc?RI-mediated signaling pathways demonstrated that emodin inhibited the phosphorylation of Syk and multiple downstream signaling processes including mobilization of intracellular Ca²⁺ and activation of the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and NF-κB pathways. When administered orally, emodin attenuated the mast cell-dependent passive anaphylactic reaction in IgE-sensitized mice. Thus, emodin inhibits mast cell activation and thereby the anaphylactic reaction through suppression of the receptor-proximal Syk-dependent signaling pathways. Therefore, emodin might provide a basis for development of a novel anti-allergic drug.     

Deficiency of SHP1 leads to sustained and increased ERK activation in mast cells, thereby inhibiting IL-3-dependent proliferation and cell death

SHP-1 plays an important role for the regulation of signaling from various hematopoietic cell receptors. In this study, we examined IL-3-induced cell proliferation and IL-3 depletion-induced apoptosis in bone marrow-derived mast cells (BMMC) established from motheaten (me) that lack SHP-1 expression, viable motheaten (me^v) expressing phosphatase-deficient SHP-1, and wild-type (WT) mice. When BMMC were stimulated with IL-3, increased ERK activation was evident in resting state and sustained in me-BMMC relative to WT-BMMC. ERK is known to be involved in the regulation of cell proliferation and apoptosis in some cells. In accordance with sustained ERK activation, apoptosis was decreased in me- and me^v-BMMC compared with WT-BMMC. In contrast to the predicted role of ERK as a pro-survival molecule, IL-3-induced cell proliferation was much lower in me- and me^v-BMMC than WT-BMMC. Stimulation with lower concentration of IL-3 or addition of PD98059, a MEK inhibitor, to the culture resulted in the suppression of decreased apoptosis and cell proliferation in me- and me^v-BMMC. Collectively, these results suggest that SHP-1 positively regulates IL-3-dependent mast cell proliferation and apoptosis by inhibiting ERK activity through its phosphatase activity. Furthermore, our results indicate that ERK would act as a negative regulator for cell proliferation and induce apoptosis when its activity is highly increased.     

Synergistic enhancement of ectopic bone formation by supplementation of freshly isolated marrow cells with purified MSC in collagen–chitosan hydrogel microbeads

ABSTRACT

Purpose: Bone marrow-derived mesenchymal stem cells (MSC) can differentiate osteogenic lineages, but their tissue regeneration ability is inconsistent. The bone marrow mononuclear cell (BMMC) fraction of adult bone marrow contains a variety of progenitor cells that may potentiate tissue regeneration. This study examined the utility of BMMC, both alone and in combination with purified MSC, as a cell source for bone regeneration. Methods: Fresh BMMC, culture-expanded MSC, and a combination of BMMC and MSC were encapsulated in collagen–chitosan hydrogel microbeads for pre-culture and minimally invasive delivery. Microbeads were cultured in growth medium for 3 days, and then in either growth or osteogenic medium for 17 days prior to subcutaneous injection in the rat dorsum. Results: MSC remained viable in microbeads over 17 days in pre-culture, while some of the BMMC fraction were nonviable. After 5 weeks of implantation, microCT and histology showed that supplementation of BMMC with MSC produced a strong synergistic effect on the volume of ectopic bone formation, compared to either cell source alone. Microbeads containing only fresh BMMC or only cultured MSC maintained in osteogenic medium resulted in more bone formation than their counterparts cultured in growth medium. Histological staining showed evidence of residual microbead matrix in undifferentiated samples and indications of more advanced tissue remodeling in differentiated samples. Conclusions: These data suggest that components of the BMMC fraction can act synergistically with predifferentiated MSC to potentiate ectopic bone formation. The microbead system may have utility in delivering desired cell populations in bone regeneration applications.     

Utilizing Fcepsilon-Bak chimeric protein for studying IgE-FcepsilonRI interactions

We previously constructed a pro-apoptotic Fcε-Bak chimeric protein, targeted against cells expressing the IgE high affinity receptor (FcεRI). We demonstrated that the chimeric protein is internalized by target mast cells and kills them. These results, which constitute a promising basis for applying this approach to antiallergic therapy, raise some theoretical questions with respect to two major issues: (a) is the monomeric Fcε-Bak–FcεRI complex able to undergo endocytosis, and (b) does the receptor binding domain of human IgE (Fcε) react with rodent FcεRI? In an attempt to answer these questions, we have now thoroughly investigate the interaction of human (h) and mouse (m) Fcε-Bak with FcεRI-positive cells. Using established cultures of rodent and human origin, as well as a primary mouse mast cell culture, we demonstrate that binding of the chimeric protein to the membrane is followed by quick endocytosis, leading to the apoptosis of specific cells. We also confirm that this interaction depends on FcεRI and not on other IgE receptors. We found that the effect of Fcε-Bak on the cells depends on the level of surface FcεRI expression, but not on the origin of the target cells or of the Fcε moiety. We suggest that endocytosis of the monomeric Fcε-Bak–FcεRI complex results from the inability of Fcε-Bak to transduce signals, characteristic of the monomeric IgE–FcεRI complex due to the absence of the variable portion of the IgE molecule. Our results also indicate that at least the Fcε fragment of human IgE is able to interact with both human and rodent FcεRI.