Effects of recombinant human H-subunit and L-subunit ferritins on in vitro growth of human granulocyte-monocyte progenitors.

We have studied the influence of purified recombinant human H-subunit (rHF, acidic) and L-subunit (rLF, basic) ferritins on in vitro colony formation by normal human granulocyte-macrophage progenitor cells (CFU-GM). Whereas rLF had no significant effect, rHF produced significant decrease in colony formation: mean inhibition of CFU-GM was 38% +/- 13% at 10(-8) M and 22% +/- 13% at 10(-9) M. The inhibitory activity of rHF was lost at 10(-10) M, and was inactivated with a monoclonal antibody recognizing the H subunit, but not with a monoclonal antibody recognizing the L subunit. Although H-type isoferritins were found in normal and leukaemic cells, their concentration in peripheral blood plasma and bone marrow plasma from normal subjects and patients with different haematological disorders including acute leukaemia were 10(-11) M or lower, i.e. levels showing no activity in vitro. We conclude that: (i) acidic H-subunit-rich isoferritins have inhibitory effects on in vitro growth of granulocyte-macrophage progenitors; (ii) levels of these isoferritins in peripheral blood and bone marrow plasma are 2-3 orders of magnitude lower than the effective concentrations in vitro, indicating that these molecules do not behave as circulating regulatory or suppressive factors in vivo.

A simple method of obtaining monocytes in suspension.

Relatively pure monocyte suspensions may be obtained by density flotation after simple conditioning of leukocytes. This consists of incubating blood or buffy coat cells in plasma containing NaCl at a higher than physiological concentration. As a result the density of granulocytes and lymphocytes increases greatly, but the density of monocytes changes very little. It is possible with one centrifugation on a slightly modified density gradient to obtain 90% pure monocyte suspensions with 55% yield. More than 95% purity but lower yield is obtained simply by increasing the NaCl concentration. The membrane characteristics of monocytes and their capacity to function normally remain unaltered by this treatment and cells so obtained develop in culture into macrophages normally. This method is much cheaper and simpler than others described, and is applicable equally to either small or large numbers of leukocytes.