Synthesis and release of erythroid colony- and burst-potentiating activities by purified populations of murine peritoneal macrophages

Abstract

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.

Human erythroid burst-forming units. Growth in vitro is dependent on monocytes, but not T lymphocytes.

“…These experiments and previous reports of BPA in media conditioned by monocytes or macrophages (7,8,11,35) suggest that monocytes are capable of producing all the BPA or other factors necessary for optimal proliferation and differentiation of human BFU-E in vitro. However, the possibility that T lymphocytes also are capable of producing small quantities of BPA has not been excluded.…”

“…Burst-promoting activity (BPA), which is a proposed regulator of the proliferation and initial stages of differentiation of BFU-E, has been detected in media conditioned by unstimulated, mitogenstimulated, or antigen-stimulated unfractionated mononuclear cells (4-8, 10, 15-18), monocytes or macrophages (7,8,11,34), or T lymphocytes (7,9,10,12,15).…”

“…Several investigators have provided evidence for the existence of another regulatory protein for BFU-E in vitro, which has been termed burst-promoting activity (4,6,7), burst-feeder activity (8), erythroid-potentiating activity (9), or lymphocyte mitogenic factor (10). Regulators of murine BFU-E have been reported to be derived from unfractionated, mitogen-stimulated spleen cells (4), plastic-adherent radioresistant bone marrow mononuclear cells (8), or adherent mouse peritoneal cells (macrophages) (11). There has been a great deal of controversy regarding the cellular source of regulators of human BFU-E.…”

“…Addition of as many as 2 x 105 T cells but no monocytes resulted in stimulation to only 34% of expected BFU-E growth. Addition of 2 x 104 T cells, which alone did not affect BFU-E growth, could augment significantly the stimulatory effect of [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] x 103 monocytes on BFU-E growth. Thus, monocytes alone appear to be capable of stimulating BFU-E growth in vitro in the presence of erythropoietin.…”

T Lymphocytes, Monocytes and Erythropoiesis Disorders in Chronic Renal Failure

“…Recently, some studies showed that the erythroid co lony (BFU-E) growth is not regulated necessarily by erythropoietin, but by cells such as T lymphocytes or monocytes and BFU-E interaction [4][5][6].…”