Monocyte requirement for mitogen-induced aggregation of human peripheral mononuclear leukocytes in vitro

Formation of distinct multicellular aggregates is one of the phenomena associated with activation of quiescent human mononuclear leukocytes in vitro. Aggregate formation involves active cell motility and enhances cell-cell interactions required for an optimal proliferative response of T-cells stimulated with agents like phytohemagglutinin. We have developed an assay to quantitate the rate at which motile cells form aggregates on a flat surface. This assay follows the time rate of deviation of cells in undisturbed culture away from an initial random distribution using an "aggregation index." We used this assay to establish minimal culturing conditions required to observe an aggregation response for a partially purified mononuclear leukocyte population. We also studied the ability to aggregate of various subpopulations enriched for T- and B-lymphocytes and monocytes and found evidence for a monocyte requirement for lymphocyte aggregation. In a second assay, we followed the rate of entry of esterase positive monocytes into aggregates and compared this to the rate of entry of mononuclear cells in toto. We found that monocytes are preferentially associated with non-esterase positive cells within one hour of PHA stimulation. The results support the conclusion that monocytes play a central role in directing the motility of human T-lymphocytes leading to their aggregation response in tissue culture.