Substance P Stimulates Both NK-1R And FcεR I α-dependent Degranulation Of Cultured Murine Mast Cells

Objective:Here we were first to investigate the effect of interleukin-4(IL-4) stimulation on the expression of FcεRⅠα and NK-1R on mature mast cells cultured and differentiated from mouse bone marrow stem cells, and then further to study if these mast cells also respond to substance P(SP) both in an FcεRⅠα- and NK-1R- dependent manner.Methods:1. Bone marrow cells were aseptically flushed from Balb/c mouse femurs, followed by culture with stem cell factor(SCF 100ng/ml), IL-3(15ng/ml) and IL-4(0,10,15,20 and25ng/ml, respectively). The culture medium was changed once a week. The morphological changes of culture cells were observed under inverted microscope.2. After four weeks’ culture, the cells were collected and appraised by toluidine blue staining and flow cytometry. The expressions of surface CD117, FcεRⅠα and NK-1R on these cells were detected by flow cytometry and Western Blotting.3. Bone marrow mast cells were activated with SP(0,0.01,0.1,1 and 10μg/ml,respectively) for 30 min. The histamine released into the supernatant and stored in the protoplasm was quantified by enzyme linked immunosorbent assay(ELISA). The percentage of histamine release was calculated as a percent of total histamine content.Results:1. The four-week cultured cells were in accordance with the characteristic of mature mast cells.2. In contrast, the expressions of FcεRⅠα and NK-1R on these mast cells co-cultured with SCF, IL-3 and IL-4 were statistically higher than that co-cultured with only SCF plus IL-3 without IL-4. Bone marrow mast cells were shown to have the highest expression of FcεRⅠα and NK-1R in culture of 20ng/ml IL-4.3. Mast cells in culture of 20ng/ml IL-4 could be activated to degranulate by a lower concentration of SP(0.01μg/ml), with the release rate of histamine from mast cells showing a positive correlation with SP concentrations. On the other hand, mast cells with high expression of FcεRⅠα and little expression of NK-1R cultured with 0 ng/ml IL-4,could also be activated by a much higher concentration of SP(1μg/ml).Conclusion:SP-controlled mast cell degranulation may be mediated through both FcεRⅠα(immunologically) and NK-1R(non-Ig E mediated or non-immunologically) pathways.These results will shed new light on the final unveiling of the mast cell degranulation mechanism.