Regulation of lymphocyte-activating factor (LAF) production and secretion in P388D₁ cells: Identification of high molecular weight precursors of LAF

Phorbol myristic acetate (PMA) markedly enhanced the production and secretion of lymphocyte-activating factor (LAF) by the murine macrophage cell line, P388D₁. The stimulatory effect of PMA proceeded, in part, via activation of an existing intracellular precursor pool of LAF. This conclusion is based on the observations that 1) the initial stimulatory effect of PMA on LAF production and secretion occurred for at least 4 hr in the presence of two inhibitors of protein synthesis, cycloheximide and puromycin, and 2) higher m.w. aggregate forms of LAF, possessing differing levels of activity, were present in the sonicates of P388D₁ cells. Five species of intracellular LAF with approximate m.w. of 220,000, 50,000 to 70,000, 39,000, 26,000, and 13,000 daltons were resolved on a Sephacryl S200 column. The 50,000 and 13,000 m.w. forms exhibited the most LAF activity, the 39,000 and 26,000, m.w. species were significantly less active, and the 220,000 m.w. form was only weakly active. The 13,000 m.w. intracellular LAF exhibited the same charge heterogeneity on DEAE cellulose as the LAF obtained from the culture supernatatns of PMA-stimulated cells, suggesting that the charge heterogeneity of LAF is probably not the result of extracellular modification but a characteristic of the intracellular factor. The 50,000 m.w. species could be completely converted to the 26,000 and 13,000 m.w. forms of intracellular LAF at 37°C. The chymotrypsin-like enzyme nor was the reaction inhibited or enhanced by sodium dodecyl sulfate. The reaction was, however, strongly temperature dependent, proceeding with low efficiency at 4° or 25°C. We propose that during the initial stage of macrophage activation ('priming'), the inactive precursor form(s) of LAF is synthesized and stored. Upon exposure of the primed cells to a secondary stimulus such as PMA, the LAF precursor is converted to an active low m.w. form and secreted. This precursor activation process would thus provide a rapid and efficient mechanism for the mobilization and release of relatively large quantities of LAF by primed or activated macrophages.