Monocyte-derived recruiting activity: Kinetics of production and effects of endotoxin

Cultured monocytes release a factor, monocyte-derived recruiting activity (MRA), which stimulates fibroblasts, endothelial cells, and T lymphocytes to produce colony-stimulating activity (CSA). We studied the kinetics of MRA production using a technique in which MRA levels were measured in a two stage bioassay. We used umbilical vein endothelial cells as the MRA-responsive granulocyte-macrophage (CFU-GM)-enriched bone marrow cells (T lymphocyte- and monocyte-depleted, low density bone marrow cells) as the CSA-responsive cells. MRA stimulated a 30-fold increase in CSA production by endothelial cells. MRA production was detected in supernatants from as few as 10³ monocytes per milliliter, required the presence of fetal calf serum, and was inhibited by cycloheximide (10 to 100 μg/mL) and puromycin (10 to 50 μg/mL). Production was detectable after 24 hours of monocyte incubation, was maintained for 3 days, and fell to undetectable levels by 7 days. With the addition of bacterial endotoxin (lipopolysaccharide [LPS]) (50 μg per 10⁶ cells), MRA was detectable after only 3 hours of incubation, and levels peaked at 24 hours. Further, maximum MRA levels in the supernatants of LPS-stimulated monocytes were up to 10 times greater than peak levels in the supernatants of unstimulated monocytes. Endotoxin augmented monocyte production of MRA to a greater extent than it did CSA production, indicating that the stimulation of CSA production by endotoxin may be at least partly indirect. The responsiveness of MRA production to endotoxin in vitro is consistent with the notion that MRA may be a biologically relevant regulator of CSA production by cells of the hematopoietic microenvironment.