STIMULATORY EFFECTS OF SUBSTANCE-P ON CD34 POSITIVE CELL-PROLIFERATION AND DIFFERENTIATION IN-VITRO ARE MEDIATED BY THE MODULATION OF STROMAL CELL-FUNCTION

Substance P (SP) is a neuropeptide widely distributed in the nervous s ystem. Extensive study has shown SP stimulates production of various c ytokines by bone marrow stromal cells, although, the role of SP in hem atopoietic phenomena is still unclear. Recently, we established a huma n cloned stromal cell line, HAS303, which can support hematopoietic st em cell proliferation and differentiation in vitro. We used this cultu re system to examine the effects of SP. Expression of the mRNAs of neu rokinin (NK)-1R, NK-2R and NK-3R, specific SP receptors, on HAS303 cel ls was demonstrated by the RT-PCR. CD34(+) cells isolated from bone ma rrow were co-cultivated with HAS303 cells in the presence and absence of SP and the total hematopoietic cells and progenitors were counted e very 5 days. Introducing SP (10(-8) M) to the co-cultures significantl y increased the number of total cells and progenitors compared with co ntrol cultures. SP showed no enhancing activity on CD34(+) cells cultu red alone. SP also stimulated IL-3-dependent colony formation of whole bone marrow MNCs in a soft agar culture system, but showed no such ac tivity on isolated CD34(+) cells in this system. These observations su ggest that SP stimulated HAS303 cells, activated HAS303 cells, and sti mulated the proliferation and differentiation of CD34+ cells. Treating HAS303 cells with SP increased the intracellular Ca2(+) concentration and stimulated production of G-CSF, GM-CSF, SCF and IL-6, but not IL- 1 alpha, IL-1 beta and TNF alpha, but did not enhance proliferation. A ll these findings suggest that SP mediates hematopoietic cell prolifer ation and differentiation in vitro by activating stromal cell function .