[Experimental study on substance P in the regulation of degranulation of cultured murine mast cells].

Objective: To investigate the effect of interleukin-4 (IL-4) stimulation on the expression of FcεRⅠα and NK-1R on mature mast cells(MC) cultured and differentiated from mouse bone marrow stem cells, and then to study if these MC also respond to substance P (SP) both in FcεRⅠα and NK-1R dependent manners. Methods: Bone marrow cells were aseptically flushed from BALB/c mouse femurs into complete RPMI 1640, followed by culture with stem cell factor (SCF 100 μg/L), IL-3 (15 μg/L) and IL-4 (0, 10, 15, 20 and 25 μg/L, respectively). The culture medium was changed once a week. The morphological changes of culture cells were observed under inverted microscope. After 4 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 blot. Bone marrow MC were activated with SP (0, 0.01, 0.1, 1.0 and 10 mg/L, 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: When different concentrations of IL-4 (0, 10, 15, 20, 25 μg/L)were added into RPMI 1640, the positive rates of CD117 on MC surface were expressed as (94.8±1.3)%, (95.7±2.5)%, (94.1±1.3)%, (96.6±1.0)%, and (96.6±1.1)%, respectively, and there was no significant difference among these groups ( F =8.51, P >0.05). The positive rates of FcεRⅠα were expressed as (81.5±2.6)%, (84.2±1.8)%, (91.8±2.0)%, (91.6±1.6)%, and (93.0±2.6)%, respectively, and there was statistically increasing among these groups ( F =15.76, P <0.05). Then MC were activated by SP (0, 0.01, 0.1, 1.0, 10 mg/L), histamine from 20 μg/L IL-4 group were released (20.08±1.50)%, (32.76±2.99)%, (42.90±3.36)%、(50.21±1.29)%, (56.10±3.60)%, as similar as from 0 μg/L IL-4 were (19.37±2.02), (19.50±1.50), (21.77±1.91), (32.00±2.50), (33.56±1.25), there was significantly different when compared with each other (all P <0.05). Bone marrow MC were shown to have the highest expression of FcεRⅠα and NK-1R in culture of 20 μg/L IL-4 by the detection of Western blot, meanwhile these MC could be activated to degranulate by a lower concentration of SP (0.01 mg/L), with the release rate of histamine from MC showing a positive correlation with SP concentrations. On the other hand, MC with high expression of FcεRⅠα and little expression of NK-1R cultured with 0 μg/L IL-4, could also be activated by a much higher concentration of SP (1.0 mg/L). Conclusions: Bone marrow mast cells were shown to be successfully differentiated and to express NK-1R and FcεRⅠα upon co-culture with SCF and IL-3 or SCF, IL-3 and IL-4.When IL-4 was added into RPMI 1640, bone marrow MC could highly produce FcεRⅠα and NK-1R, thus building a better model of MC degranulation regulated by SP. And SP-controlled MC degranulation may be mediated through both FcεRⅠα (immunologically) and NK-1R (non-IgE mediated or non-immunologically) pathway.