IL-3 IS PRODUCED BY NORMAL STROMA IN LONG-TERM BONE-MARROW CULTURES

Interleukin-3 (IL-3) has been shown to halve significant effects on ha emopoiesis in vitro, but early investigations of normal human long-ter m bone marrow cultures (LTBMC) have failed to demonstrate IL-3 product ion by stromal cells, either by Northern blotting for mRNA, or assayin g for bioactivity in culture supernatants, One recent report, using re verse transcription-polymerase chain reaction (RT-PCR), demonstrated I L-3 expression in only one of eight cultures. We have developed a sens itive bioassay for the detection of IL-3 production from normal stroma in LTBMC, LTBMC were grown until confluent, irradiated, and stroma ha rvested by trypsinization to yield single-cell suspensions. These cell s were then cocultured with target bone marrow mononuclear cells (BMMC ), or CD34(+) cells in clonogenic assays, either in the presence or ab sence of anti-IL-3 neutralizing antibodies, We have demonstrated IL-3 production in 32/34 cases, In addition, by separating stroma from targ et cells using cell culture inserts, we have shown that direct stroma: stem cell contact is not necessary for colony growth, suggesting that IL-3 diffuses into the supernatant, However, when supernatants from LT BMC were assayed by enzyme-linked immunoassay (ELISA), no IL-3 was det ected. This suggests that IL-3 is probably produced at low levels and has a short-range interaction. Stroma production of IL-3 was confirmed by the detection by RT-PCR of IL-3 mRNA in 3/3 cases. The simultaneou s detection of CD2 mRNA demonstrated that T cells are part of the bone marrow stroma, It is therefore possible and probably likely that thes e cells are the source of IL-3.