Lactoferrin (LF), the iron-binding protein present in the specific granules of mature granulocytes has been identified as colony inhibitory factor (CIF) which suppresses granulocyte--macrophage colony stimulating activity (CSA) production by monocytes and macrophages in vitro and rebound granulopoiesis in vivo. Separation of LF and CIF by isoelectric focusing confirmed that the regions of inhibitory activity corresponded in both to a pH of congruent to 6.5. In addition, the purified immunoglobulin fraction of rabbit anti-human LF antiserum, but not rabbit anti-transferrin (TF), inactivated the capacity of LF and CIF to inhibit CSA production, an effect blocked by prior incubation of anti-LF with neutralizing concentrations of LF. Suppression of CSA production correlated with the iron-saturation of LF; APO-LF (depleted of iron) was only active concentrations greater than 10(-7) M, native LF (8% iron saturated) was active at 10(-15) M, and fully iron-saturated LF inhibited at 10(-17) M. Suppression of CSA production occurred within a 1/2-h preincubation period with human blood monocytes but was reversed by bacterial lipopolysaccharide (LPS). This reversal was dependent on the relative concentrations of LF to LPS. Serum TF, a biochemically similar iron-binding protein which is antigenically distinct from LF, was only minimally active at concentrations greater than 10(-6) M. LF did not inhibit exogenously stimulated human granylocyte and macrophage colony-forming cells or erythropoietin-dependent human or murine erythroid colony- or erythroid burst-forming cells. Microgram quantities of LF acted in vivo to inhibit rebound granulopoiesis and CSA production in CD1 and C57Bl/6 mice pretreated with cyclophosphamide. These results strongly implicate LF as a physiological regulator of granulopoiesis.

WEHI-3 cell-conditioned medium with the capacity to stimulate megakaryocyte colony formation was separated by Sephadex G-150 column chromatography. The development of colonies containing megakaryocytes was observed only when mixing experiments were performed. Individual fractions did not support megakaryocyte colony growth. The two factors in WEHI-3 CM required for megakaryocyte colony growth had apparent average molecular weights of 35,000 daltons (megakaryocyte CSF) and 100,000 daltons (megakaryocyte potentiator). The results were confirmed in serum-free conditions in which colonies were directly identified in the cultures by acetylcholinesterase staining. Two growth factors may be necessary for the genesis of megakaryocytic colonies.

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The intramembrane organization of the plasma membranes of nonmalignant cells in culture has been compared by freeze-fracturing with that of virally-transformed malignant cells. No dramatic differences are present in the distribution of intramembrane particles in the plasma membranes of these cells when the cells are examined without fixation or with mild fixation (glutaraldehyde treatment) prior to freezing. However, a redistribution of intramembrane particles into aggregates occurs in the membranes of nontransformed cells after treatment with glycerol. The aggregation of particles is extensive in normal chick embryo fibroblasts, and less extensive in mouse 3T3 cells. The glycerol-induced particle redistribution is not inhibited at 40, but it is inhibited by pretreatment with 2.5% glutaraldehyde. A significant number of the cells remain viable after the glycerol treatment, and the process is reversible. Particle aggregation does not appear to be related to either growth rate or cell density. Transformed Rous sarcoma virus/chick embryo fibroblasts and simian virus 40/3T3 cells have few particle aggregates after glycerol treatment. The plasma membranes of chick embryo fibroblasts transformed with a mutant of Rous sarcoma virus (TS-68) that is temperature sensitive for transformation, have few particle aggregates when grown at the permissive temperature (37°). Extremely prominent particle aggregates are present in the plasma membranes ofcells grown at the nonpermissive temperature (410). These observations indicate that there is an alteration in the plasma membrane associated with viral transformation which is related to a glycerol-sensitive mechanism that controls the distribution of intramembrane particles. Malignant transformation of cells in culture has been associated with several changes, such as increased protease production (1), biochemical changes in cell surface components (2-6), increased lectin agglutinability (6, 7), and cell shape changes (8, 9). All of these changes may be directly or indirectly related to modification of the cell surface membrane.Recently, Scott et al. (10,11) reported that differences exist in the internal structure of the membranes of normal and transformed cells. With the freeze-fracture technique, these workers found that at high cell densities the intramembrane particles were aggregated in the membranes of normal cells, while at similar densities, the intramembrane particles were dispersed in transformed cell plasma membranes. Several questions were raised from the data in this earlier study. (i) Were intramembrane particle aggregates a characteristic feature of nontransformed cell membranes, or were they induced by the glycerol treatment that was used for cryoprotection during the preparation of the cells? In other studies on intact cells, it has been demonstrated that glycerAbbreviations: CEF, chick embryo fibroblasts; RSV, Rous sarcoma virus; TS-68, temperature-sensitive Rous sarcoma virus; 3T3, 3T3 mouse cell line; SV40, simian virus 40. ol, as well as dimethyl s...