The Journal of Immunology, Vol 142, Issue 2 543-548, Copyright © 1989 by American Association of Immunologists

ARTICLES

Identification of a macrophage-activating factor in granules of the RNK large granular lymphocyte leukemia

E Roussel and AH Greenberg
Department of Immunology, University of Manitoba, Winnipeg, Canada.

Recent work from our laboratory has shown that NK cells rapidly release preformed factor(s) that stimulate monocyte oxidative metabolism and microbicidal activity. We have hypothesized that such factors could also activate macrophage (M phi) tumor lysis and might be stored in the cytoplasmic granules. Granules were isolated from the RNK large granular lymphocyte leukemias by nitrogen cavitation and Percoll fractionation of the cell homogenate. Utilizing CSF-1 differentiated murine bone marrow-derived M phi and P815 tumor target cells, a M phi- activating factor (MAF) was found. The MAF activity was identified in two peaks, the first was coincident with dense granule enzymes and was 60 times more concentrated per mg protein than a second peak in the cytosol fractions. Solubilization in 2 M NaCl was necessary to recover activity from both peaks. Granule NK-MAF required the simultaneous presence of LPS in order to induce tumoricidal activity. Kinetics of NK- MAF activation peaked after 12 h of exposure. The NK-MAF was short lived in the solubilized granules; however, its heat resistance allowed us to prepare enriched and stable preparations. Treatment of NK-MAF with pepsin but not trypsin completely abrogated its activity. The NK- MAF passed through an ultrafiltration membrane with a nominal cut-off of 10 kDa. This work indicates that NK cell granules contain a small heat-stable peptide capable of activating M phi tumoricidal activity.