To characterize the production of
stem cell factor (SCF, the
ligand for the
c-kit receptor protein) and its regulation by inflammatory
cytokines and
glucocorticoids, primary marrow stromal fibroblasts were isolated from normal individuals and two patients with
Diamond-Blackfan anemia. Unstimulated normal marrow stromal fibroblasts constitutively expressed a low level of SCF
mRNA (9 +/- 2 copies/cell [mean +/- SEM]), continually secreted soluble SCF into the supernatant of 1- to 5-day-old cultures (0.16 +/- 0.02 to 0.73 +/- 0.04 ng/mL per 10(6) cells, respectively), and expressed membrane-bound SCF. Stimulation with
interleukin-1 beta (IL-1 beta) only modestly increased SCF
mRNA levels, soluble SCF production at 24 hours, and membrane-bound SCF. In comparison,
hydrocortisone or
tumor necrosis factor alpha (
TNF-alpha) exposure increased SCF
mRNA levels 3.5- to four-fold above controls, but with different kinetics. The peak
TNF-alpha effect was at 6 hours, with return to near 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 detected during 1 to 5 days of
hydrocortisone exposure (0.27 +/- 0.03 to 1.10 +/- 0.08 ng/mL per 10(6) cells), while
TNF-alpha stimulation modestly increased the production of soluble SCF in 24-hour cultures only. Unstimulated normal marrow fibroblasts expressed predominantly the long species of alternatively spliced SCF
mRNA, and the relative amounts of long and short mRNAs did not change after stimulation with
IL-1 beta,
hydrocortisone, or
TNF-alpha. SCF production by marrow stromal fibroblasts from a symptomatic patient with
Diamond-Blackfan anemia was equivalent to simultaneously studied normal marrow fibroblasts. In contrast, marrow fibroblasts from a
Diamond-Blackfan anemia patient in untreated hematologic remission constitutively expressed high levels of SCF
mRNA (21 +/- 4 copies/cell) and soluble
protein (0.40 ng/mL per 10(6) cells at 24 hours). Together, these observations suggest that SCF is constitutively produced by fibroblasts in the human marrow microenvironment and that
hydrocortisone induces a modest but sustained increase in SCF gene expression and
protein production, compared to only a transient increase induced by
TNF-alpha. In addition, these findings support the hypothesis that endogenous or
corticosteroid-induced increases in the production of SCF could play a physiologic role in the clinical improvement of congenital
anemia.