MIP-1α and TGF-β Production in CD34+ Progenitor–Stromal Cell Coculture Systems: Effects of Progenitor Isolation Method and Cell–Cell Contact

Macrophage inflammatory protein-1α (MIP-1α) is a C–C chemokine which has antiproliferative effects on early hematopoietic progenitors and stimulatory effects on later progenitors. It also possesses chemotactic and activating properties for monocytes, macrophages, and T-cells. CD34+ progenitors isolated utilizing an avidin–biotin immunoadsorption column produced significant amounts of MIP-1α from 24 h onward when cultured in medium with 10% fetal calf serum (>200 pg/ml). Such production persisted through 96 h of culture and was greater when such progenitors were cocultured with a preformed marrow stromal layer (4000 pg/ml at 24 h). The production of MIP-1α declined over time of coculture with stromal layers, and stromal layers themselves produced minimal MIP-1α as detected by ELISA: <100 pg/ml. In contrast, CD34+ cells isolated by flow cytometry or by magnetic bead adsorption produced minimal MIP-1α (0–30 pg/ml). MIP-1α production also increased when cells isolated by these two methods were cocultured with stromal layers. The difference in MIP-1α production could not be accounted for by differences in purity of the CD34+ population between isolation methods nor on the basis of monocytic or lymphocytic contamination as assessed by the presence of CD14 or CD3 positive cells. CD34+ cells isolated by immune adsorption had increased expression of endothelial and mesenchymal associated antigens, however, suggesting that this subpopulation might account for the MIP-1α production observed. Freshly isolated CD34+ cells expressed MIP-1α message as assessed by RT–PCR and by in situ hybridization. Coculture of CD34+ cells isolated by any means with stromal cells increased transforming growth factor-β (TGF-β) production, in this case by the stromal layer itself. Both MIP-1α and TGF-β have been found to influence cell cycle status and proliferation status of early hematopoietic progenitors, and both have potential effects on accessory cell function. These studies indicate that progenitor–stromal cell interactions may influence local cytokine output, thus potentially influencing progenitor cycling status and accessory cell activation. The method of isolation of CD34+ progenitors may influence secretion of certain cytokines and chemokines.