Human granulopoiesis in vitro--medium-dependent growth regulation by a granulocyte-derived inhibitor

A number of reports have indicated that mature blood granulocytes produce regulators that inhibit proliferation of progenitor cells in the bone marrow. However, this concept of negative feedback of granulopoiesis is still controversial. To examine whether conflicting results may depend upon the experimental set-up, we have compared colony formation by human bone marrow cells in different growth media. Unmodified McCoy's medium, which in feeder layer cultures supports the formation of large numbers of colonies, was a poor growth medium in cultures supplied with crude or recombinant colony stimulating factor (CSF). The colony formation improved when the medium was supplemented with defined additives. In CMRL 1066 cultures, granulocyte extract (GRE) consistently caused a strong inhibition of colony formation. In contrast, with unmodified McCoy's medium, granulocyte extract enhanced colony formation in a dose-dependent manner. The enhancing effect of granulocyte extract coincided with low colony numbers in the control cultures. The stimulatory effect of granulocyte extract in McCoy's medium, switched to strong inhibition when thymidine, a component of CMRL 1066 medium, was added. The inhibitory and stimulatory activities were found in the same molecular weight fractions (30-60 kD) after gel filtration. Both modulators in granulocyte extract appeared to be independent of monocytes and T lymphocytes in the bone marrow, as shown by removal of these cells with magnetic microspheres coated with specific monoclonal antibodies. The present work shows that regulation of cell proliferation in vitro depends strongly on culture conditions, such as choice of medium. It appears that thymidine acts as co-factor for the inhibitor in granulocyte extract.