IL-3 is produced by normal stroma in long-term bone marrow cultures

Area of Science:

• Hematology
• Immunology
• Cell Biology

Background:

• Interleukin-3 (IL-3) is vital for hematopoiesis, but its production by human bone marrow stromal cells in long-term cultures (LTBMC) has been difficult to detect.
• Previous studies using Northern blotting, bioassays, and limited RT-PCR failed to consistently demonstrate IL-3 production by stromal cells.

Purpose of the Study:

• To develop a sensitive bioassay for detecting IL-3 production by normal human stromal cells in LTBMC.
• To investigate the mechanism of IL-3 action and identify the source of IL-3 within the bone marrow stroma.

Main Methods:

• Developed a sensitive bioassay using cocultures of stromal cells with target bone marrow mononuclear cells (BMMC) or CD34+ cells in clonogenic assays.
• Utilized anti-IL-3 neutralizing antibodies and cell culture inserts to assess IL-3 dependency and cell contact.
• Confirmed IL-3 mRNA expression using reverse transcription-polymerase chain reaction (RT-PCR) and detected CD2 mRNA to identify T cells.

Main Results:

• Demonstrated IL-3 production in 32/34 LTBMC cultures using the developed bioassay.
• Showed that direct stroma:stem cell contact is not necessary for colony growth, indicating IL-3 diffusion.
• Detected IL-3 mRNA in 3/3 cases, and CD2 mRNA, suggesting T cells within the stroma are a likely source of IL-3.

Conclusions:

• Normal human bone marrow stromal cells produce IL-3, detectable with a sensitive bioassay.
• IL-3 likely acts via short-range diffusion at low levels, and T cells within the bone marrow stroma are probable producers.