The Role of GM-CSF in the Generation of CD1a Positive Cells in vitro by JMML Patients

Objective: Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disorder of childhood. It was recently demonstrated that mononuclear cells from spleen, peripheral blood and bone marrow of the patients with JMML proliferate in vitro and population of the cells differentiate within 7 days to the CD1a positive dendritic cells. In the present study, the role of GM-CSF in the proliferation and differentiation of the JMML cells in vitro was investigated.
Methods: MNC from patients with JMML was cultured for 7 days in RPMI/10%FCS and 1% penicillin-streptomycin. The phenotype of the cultured cells was determined at day 7 using antibodies against Ki-67, CD1a, CD14 and CD68. GM-CSF concentration was first measured at days 1, 3, 5 and 7 in the supernatants of the cultured mononuclear cells from two patients with JMML. To study the roll of GM-CSF in JMML cultures, GM-CSF activity was neutralized using monoclonal antibody (Ab) against human GM-CSF in different concentrations (0.05 µg/ml, 0.5 µg/ml, 5 µg/ml and 10 µg/ml). The effect of neutralizing antibody was determined at day 7 in treated and untreated cultures by immunostaining using antibodies against Ki-67, CD68, CD1a and CD14. The expression of GM-CSF, GM-CSFR, TNF-α, IFN-γ and SCF was also determined at day 7 in mononuclear cells treated and untreated cultures by RT-PCR.
Findings: GM-CSF neutralization led to decrease of the total cell number. Additionally, neutralization of GM-CSF decreased in dose dependent manner the number of CD1a, CD14 and Ki-67 positive cells in JMML cultures, whereas the percentage of CD68 remained unchanged. The expression of GM-CSF, GM-CSFR, TNF-α and SCF could be demonstrated at the mRNA level in the antibody treated and untreated culture as well.
Conclusion: GM-CSF plays an important role in the proliferation and differentiation of JMML cells into the CD1a and CD14 positive cells.

Keywords
Juvenile myelomonocytic leukemia, CD1a, Dendritic cell, GM-CSF, Stem cell factor, Suspension cell culture, ELISA, Immunostaining, RT-PCR