Characterization of Lymphocyte-Activating Factor (LAF) Produced by the Macrophage Cell Line, P388D1

Abstract

In the absence of exogeneous stimulation, the murine macrophage cell line, P388D1, produced low but significant amounts of lymphocyte activating factor (LAF). When P388D1 cells were incubated with PHA-activated DBA/2 mouse or Hartley guinea pig T cells, LAF production was greatly enhanced. LAF activity was not detected in cultures of only PHA-activated T cells, or in cultures of T cells incubated with P388 cells, the parent nonmacrophage lymphoma from which the P388D1 cell line was obtained. P3888D1-derived LAF was also shown to be biologically distinct from the guinea pig lymphocyte mitogenic factor that, unlike LAF, is produced by PHA-activated T cells. These results, therefore, suggest that P388D1 cells are the cell source for most, if not all, the LAF produced in mixed cultures of P388D1 cells and T cells. The stimulatory effect of activated T cells on LAF production was dependent on cell contact, since LAF production was not enhanced when P388D1 cells and T cells were separated by a semipermeable Nuclepore membrane in a Marbrook culture vessel. T cells did not have to proliferate in order to stimulate P388D1-LAF production, but T cell viability was essential. In addition, the level of LAF activity was dependent on at least three culture variables: the time of incubation of P388D1 cells and T lymphocytes, the ratio of P388D1 cells to T lymphocytes, and the fetal calf serum concentration. Based on the results of these studies, it appears that P388D1 cells are an excellent source of LAF for both biologic and biochemical studies.