A functional subpopulation of murine B lymphocytes proliferate in semisolid agar culture in the presence of 2-mercaptoethanol to form colonies. The effects of diffusible macrophage-derived factors on this focal proliferation was investigated using a two-layer culture system which prevented macrophage-lymphocyte contact and permitted B-cell activation to be critically assessed under conditions of extremely low cell densities. Adherent peritoneal macrophages incorporated within underlayers of spleen or lymph node cell cultures potentiated both the number and size of developing B-cell colonies. These effects were most striking when low numbers of spleen or lymph node cells, or macrophage- depleted lymphoid cell suspensions were used. Thus, macrophage-depleted lymph node ceils gave rise to virtually no colonies, but colony-forming ability was restored by the presence of an optimal number of macrophages. When the number of macrophages exceeded that required for optimal stimulation, colony formation was suppressed; an effect which was largely prevented by indomethacin, an inhibitor of prostaglandin synthesis. Under these conditions, stimulation and inhibition of B-cell activation by macrophages could be dissociated, indicating that each signal is selectively controlled by individual molecules elaborated by the macrophage. With an appropriate number of macrophages required for B-cell activation, and sufficient indomethacin to inhibit the accumulation of macrophage-derived prostaglandin, B-lymphocyte clonal proliferation was a linear function of the number of B cells placed in culture. In the absence of macrophages, B-cell colony formation was potentiated by both lipopolysaccharide and intact sheep erythrocytes through a mechanism different from that of the macrophage-derived stimulatory factor. In addition to their direct stimulatory effect on B-cell proliferation, lipopolysaccharide and sheep erythrocytes were each capable of modulating the production and/or release of B-cell stimulatory and inhibitory factors by the macrophage. Parallel studies of conventional mitogen- stimulated lymphocyte cultures did not show a requirement for macrophages and confirm that the semisolid assay is uniquely suited to studies on the regulatory role of the macrophage in B-cell activation.

We investigated the effects of murine resident peritoneal macrophages on the in vitro proliferation of erythropoietin (Ep)-sensitive committed precursors colony-forming unit-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) with a two-layer cloning system of methylcellulose and semisolid agar. The addition of increasing numbers of macrophages to the agar underlayer resulted in a progressive increase in the numbers of both CFU-E and BFU-E that proliferated in the presence of Ep. CFU-E, but not BFU-E, proliferated to form colonies in the absence Qf exogenously added Ep, and this proliferation was enhanced in a dose-dependent fashion by the presence of macrophages in the underlayer. The enhancing effects of a given number of macrophages and a given concentration of Ep were greater than the sum of the individual effects of macrophages and Ep alone. This erythropoietic syngerism was more evident with BFU-E because burst formation was not seen in the absence of exogenously added Ep. Macrophage underlayers stimulated three to five times the number of erythroid bursts seen with Ep alone. Cell-free agar underlayers or agar underlayers prepared with nonadherent peritoneal cells or unseparated cells from thymus, lymph node, or spleen failed to augment Ep- dependent erythroid colony formation. No enhancement of CFU-E or BFU-E was demonstrable after depletion ofadherent cells from peritoneal cell suspensions by passage over columns of Sephadex G-10. Analysis by sedimentation velocity of peritoneal cells confirmed that the cells responsible for elaborating the erythroid-enhancing activity were macrophages on the basis of morphologic, histochemical, and functional criteria. Serum- free media conditioned by macrophages in the absence of Ep contained the erythroid-enhancing activities, which indicated that Ep was not required for the elaboration of these diffusible substances. These studies indicate that although macrophages are not obligate for the growth of erythroid precursors, they serve as an important source of diffusible factors that reduce the in vitro requirement for Ep.

Analysis of in vitro colony formation in agar cultures of foetal haemopoietic tissues of eight mammalian species has shown that granulocyte‐macrophage progenitor cells are present in foetal liver, yolk sac, marrow and spleen in numbers approaching the incidence in adult marrow. Such characteristics as buoyant density, growth rate and differentiation served to distinguish foetal from adult colony forming cells (CFCs). Cell cycle analysis performed by exposing haemopoietic cells to high doses of tritiated thymidine in vitro showed that foetal CFC proliferation in species of short gestation (rabbit, rat, mouse) approached or exceeded that observed in adult marrow. In contrast, in species of long gestation (human, monkey, calf, lamb, guinea‐pig) a period of variable duration was observed when foetal liver CFCs entered a non‐cycling G0 or blocked G1 phase. In these species foetal liver CFCs were found to be proliferating actively early in gestation and following the non‐cycling phase again re‐entered a proliferative state associated with onset of active granulopoiesis in foetal marrow and possible migration of CFC from liver to marrow. These results indicate the existence of granulocyte‐macrophage progenitor populations displaying foetal characteristics and adapted to particular stages of haemopoietic development, a situation which closely parallels that reported for erythropoiesis.

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