IL‐3 is produced by normal stroma in leng‐term bone marrow cultures

Abstract

Interleukin-3 (IL-3) has been shown to have significant effects on haemopoiesis in vitro, but early investigations of normal human long-term bone marrow cultures (LTBMC) have failed to demonstrate IL-3 production by stromal cells, either by Northern blotting for mRNA, or assaying for bioactivity in culture supernatants. One recent report, using reverse transcription-polymerase chain reaction (RT-PCR), demonstrated IL-3 expression in only one of eight cultures. We have developed a sensitive bioassay for the detection of IL-3 production from normal stroma in LTBMC. LTBMC were grown until confluent, irradiated, and stroma harvested by trypsinization to yield single-cell suspensions. These cells were then cocultured with target bone marrow mononuclear cells (BMMC), or CD34+ cells in clonogenic assays, either in the presence or absence of anti-IL-3 neutralizing antibodies. We have demonstrated IL-3 production in 32/34 cases. In addition, by separating stroma from target 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 LTBMC were assayed by enzyme-linked immunoassay (ELISA), no IL-3 was detected. 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 simultaneous detection of CD2 mRNA demonstrated that T cells are part of the bone marrow stroma. It is therefore possible and probably likely that these cells are the source of IL-3.

Cytokines active on granulomonopoiesis: release and consumption by human marrow myeloid stromal cells

“…This result is in agreement with the RT-PCR studies, where no transcripts were detected even after two rounds of detection . IL-3 was not synthesized by stromal cells, confirming many previous reports, with the exception of that of Gibson et al (1995) who found biologically active IL-3 in adherent layers of LTBMC, which may be due to the presence of remaining T cells. Consumption of IL-3 by stromal cells from CDCL was specific for this cytokine since it was not found for TGFb1 (this study), IL-4 (data not shown) and was not due to protein degradation by stroma (data not shown).…”

MMCP-8, the first lineage-specific differentiation marker for mouse basophils. Elevated numbers of potent IL-4-producing and MMCP-8-positive cells in spleens of malaria-infected mice

“…The exact source of the IL-3 is not known but an attractive possibility is that at least a fraction of it is produced by non-hematopoietic cells in the surrounding tissue. IL-3 is, for example, known to be produced by stromal cells in the bone marrow [26]. Taken together, our data give additional support for the view that our immune system is more complex than previously anticipated, gaining stability and plasticity from a highly intricate web of interactions.…”

Enhancement of terminal B lymphocyte differentiationin vitro by fibroblast-like stromal cells from human spleen

“…We can offer no valid explanation for this discrepancy at present. It is feasible, however, that IL-3 or IL-3-like activity is produced in very small amounts by stromal cells, a possibility already shown by Gibson et al [23] in cultures of bone marrow stroma. Neutralizing antibodies against IL-10 also failed to exert any effect showing that IL-10 was not involved in our experimental system.…”