Effect Of SCF Signal On IL-13 Production And Migration Of Mast Cells

Background:Mast cells originate from bone marrow hematopoietic cell and their precursors become mature in tissues. Mast cells are involved in allergy response(including asthma) as effectors. On the other hand, it can regulate a few biological processes in vivo though expressing costimulatory molecular and producing cytokines. Mast cells can release histsmine and other inflammatory mediators by cross-linked high-affility FcεRI receptor. So, mast cells are involved in early-phase reactions of allergic response. Meanwhile, mast cells are also involved in late-phase reactions in allergic response though the recruitment of eosinophils and neutrophil to local tissures by releasing chemokines such as eotaxin and IL-8.Stem cell factors(SCF), also known as mast cell growth factor, is a cytokine produced by diverse cells, including fibroblasts, epithelial cells, mast cells, eosinophil and airway smooth muscle cells. SCF is an important growth factor in vivo and it can promote hematopoietic precursor cells, melanoma cells and germ cells to proliferate,migrate, differentiate and survive. SCF signal also plays a key role on the growth,survival, adhesion, activation and other biological activity in mast cells. Now, studies have shown that the content of SCF is increased in serum and sputum of patients with asthma, but the relationship between SCF and asthma is unclear.Bronchial asthma(asthma) is one of the most prevalence chronic respiratory system diseases and its incidence is on the rise in recent years. Asthma is a chronic airway inflammation which is featured by infiltration of eosinophils, neutrophils, mast cells, T cells and so on and airway hyperresponsiveness and remodeling are its main characteristic. Some studies showed that the degree of severity of asthma symptoms is related with mast cell numbers and activation in airway. It is well-known that the development of asthma is associated with IL-13, a typical Th2 cytokine. IL-13 ismainly produced by activated T cells, mast cells and eosinophil cells. It can result in airway hyperreactivity and remodeling though promoting fibroblaste transform into myofibroblasts, collagen deposition and airway smooth muscle proliferation. Recent studies suggest that SCF is related to cockroach allergen-mediated airway remodeling,but detailed mechanism is unclear. Since mast cells play a vital role in the occurrence and development of asthma. In order to illustrate the relationship between SCF and asthma, it is necessary to study the effect of SCF signal on IL-13 generation and migration of mast cell.Objective : To investigate the effect of SCF singal on IL-13 production and migration in mast cells.Methods:1. The expression of c-Kit receptor in P815 cells was detected by using Western blot and Flow cytometry.2. The secretion of IL-13 was detected by ELISA after P815 cells were stimulated by various concentrations of SCF at different times.3. ELISA was used to test the secretion of IL-13 in P815 cells which were stimulated by SCF(50ng/ml) with or without JSI-124(100n M), U0126(10μM), Wortmannin(1μM), PDTC(50μM) and H-89(20μM) pretreatment for 30 minutes.4. Western blot was used to detect the phpsphorylation of signal transduction protein Erk in P815 cells which were stimulated by SCF(50ng/ml) at different times(15min,30 min, 60min).5. EMSA was used to detect the activation of nuclear transcription factor CREB in P815 cells which were stimulated by SCF(50ng/ml) for 1h.6. The expression of mi R-200 c, mi R-194, mi R-34b-5p, mi R-449 b, mi R-200 b,mi R-98, mi R-410, mi R-429, mi R-362-3p, mi R-34 a, mi R-34 c and mi R-449 c were tested by fluorescent quantitation PCR after P815 cells were stimulated by SCF(50ng/ml) for 6h.7. Transwell assay was used to observe the changes of the migration cell numbers in P815 cells which was stimulated by SCF(10ng/ml) stimulus alone or with Anti-CCL2(1ng/ml) antibody or with CCL2 isotype antibody(1ng/ml) pretreatment for 2h.Results:1. Mast cells P815 expressed c-Kit. Almost all of the P815 cells express c-Kit surface receptor.2. ELISA results show that SCF(1-100ng/ml) can induce IL-13 release from P815 cells following 6h incubation(p<0.01). This effect is most obvious when the concentration of SCF is in the range of 10-50ng/ml. P815 cells were stimulated by SCF(50ng/ml) at different times(6h, 12 h, 24 h, 48 h, 72h), IL-13 production was significantly increased(p<0.01). In addition, IL-6 secretion was increased(p<0.05),whereas IFN-γ and IL-10 secretion was decreased(p<0.05) when P815 cells were stimulated by SCF(50ng/ml) for 6h.3. Compared with the SCF(50ng/ml) single stimulation group, there is no significant difference about IL-13 production in JSI-124 + SCF group, Wortmannin + SCF group and PDTC + SCF group P815 cells. But U0126+SCF group and H-89+SCF group P815 cells had significantly decreased IL-13(p<0.01).4. Wstern blot results show that Erk1/2 phosphorylation bands are weak in unstimulated P815 cells. But the phosphorylated Erk1/2 bands brightness increased after SCF(50ng/ml) stimulation for 15 min, 30 min peaked. However, there is a decreasing trend in 60 min stimulation group.5. CREB hysteresis band is weak in unstimulated P815 cells. But this pattern band is obvious after P815 cells were stimulated by SCF(50ng/ml) for 1h.6. When P815 cells were stimulated by SCF(50ng/ml) for 6h, the expression of mi R-200 c, mi R-34b-5p, and mi R-449 b were increased and the expression of mi R-98,mi R-200 b, mi R-410, mi R-429, mi R-362-3p, mi R-34 a, mi R-34 c, mi R-194 and mi R-449 c were decreased. Moreover, mi R-449 c abatement is most obvious among them.7. Compared with unstimulated group, migration cell numbers were significantly increased when P815 cells were stimulated by SCF(10ng/ml) at different times(3h,6h, 12 h, 24h)(p<0.01). Compared with the SCF(10ng/ml) single stimulation group,the numbers of migration cells were decreased in Anti-CCL2 + SCF(10ng/ml) group(p<0.01).Conclusion:1. Almost all of the P815 cells express c-Kit receptor on their surface.2. SCF induces P815 cells to release IL-13 though activating MEK-ERK-CREB signaling pathway.3. SCF accelerate the migration of P815 cells and this promotion effect of SCF is concerned with CCL2.