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IL‐3 is produced by normal stroma in leng‐term bone marrow cultures
Author(s) -
GIBSON F. M.,
SCOPES J.,
DALY S.,
RIZZO S.,
BALL S. E.,
GORDONSMITH E. C.
Publication year - 1995
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.1995.tb05578.x
Subject(s) - stromal cell , bone marrow , stroma , microbiology and biotechnology , haematopoiesis , clonogenic assay , biology , cell culture , trypsinization , stem cell , blot , chemistry , immunology , biochemistry , cancer research , immunohistochemistry , enzyme , trypsin , genetics , gene
Summary. 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 LL‐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 4 cells in clonogenic assays, either in the presence or absence of anti‐IL‐3 neutralizing antibodies. We have demonstrated IL‐3 production in 32β4 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 LL‐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 3β 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.