STEM-CELL FACTOR PRODUCTION BY HUMAN MARROW STROMAL FIBROBLASTS

To characterize the production of stem cell factor (SCF, the ligand fo r the c-kit receptor protein) and its regulation by inflammatory cytok ines and glucocorticoids, primary marrow stromal fibroblasts were isol ated from normal individuals and two patients with Diamond-Blackfan an emia. Unstimulated normal marrow stromal fibroblasts constitutively ex pressed a low level of SCF mRNA (9 +/- 2 copies/cell [mean +/- SEM]), continually secreted soluble SCF into the supernatant of 1- to 5-day-o ld cultures (0.16 +/- 0.02 to 0.73 +/- 0.04 ng/mL per 10(6) cells, res pectively), and expressed membrane-bound SCF. Stimulation with interle ukin-1 beta (IL-1B) only modestly increased SCF mRNA levels, soluble S CF production at 24 hours, and membrane-bound SCF. In comparison, hydr ocortisone or tumor necrosis factor alpha (TNF-alpha) exposure increas ed SCF mRNA levels 3.5- to four-fold above controls, but with differen t kinetics. The peak TNF-alpha effect was at 6 hours, with return to n ear control levels at 24 hours, whereas hydrocortisone induced maximal mRNA increases at 12 to 18 hours, and the levels remained high at 24 hours. Similarly, a sustained increase in soluble SCF production was d etected during 1 to 5 days of hydrocortisone exposure (0.27 +/- 0.03 t o 1.10 +/- 0.08 ng/mL per 10(6) cells), while TNF-alpha stimulation mo destly increased the production of soluble SCF in 24-hour cultures onl y. Unstimulated normal marrow fibroblasts expressed predominantly the long species of alternatively spliced SCF mRNA, and the relative amoun ts of long and short mRNAs did not change after stimulation with IL-1 beta, hydrocortisone, or TNF-alpha. SCF production by marrow stromal f ibroblasts from a symptomatic patient with Diamond-Blackfan anemia was equivalent to simultaneously studied normal marrow fibroblasts. In co ntrast, marrow fibroblasts from a Diamond-Blackfan anemia patient in u ntreated hematologic remission constitutively expressed high levels of SCF mRNA (21 +/- 4 copies/cell) and soluble protein (0.40 ng/mL per 1 0(6) cells at 24 hours). Together, these observations suggest that SCF is constitutively produced by fibroblasts in the human marrow microen vironment and that hydrocortisone induces a modest but sustained incre ase in SCF gene expression and protein production, compared to only a transient increase induced by TNF-alpha. In addition, these findings s upport the hypothesis that endogenous or corticosteroid-induced increa ses in the production of SCF could play a physiologic role in the clin ical improvement of congenital anemia.