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Identification of a macrophage-activating factor in granules of the RNK large granular lymphocyte leukemia
1Department of Immunology, University of Manitoba, Winnipeg, Canada.
Abstract:
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.
Insights
Natural killer (NK) cells release granule proteins that activate macrophages (M phi) to kill tumor cells. This study identified a heat-stable peptide in NK cell granules responsible for this M phi tumoricidal activity.
Area of Science:
- Immunology
- Cell Biology
Background:
- Natural killer (NK) cells release factors stimulating monocyte oxidative metabolism and microbicidal activity.
- These factors may also activate macrophage (M phi) tumor lysis and are hypothesized to be stored in cytoplasmic granules.
Purpose of the Study:
- To investigate if NK cell granules contain factors that activate macrophage tumoricidal activity.
- To characterize the nature of the macrophage-activating factor (MAF).
Main Methods:
- Isolated granules from NK cell leukemias using nitrogen cavitation and Percoll fractionation.
- Assayed MAF activity using CSF-1 differentiated murine bone marrow-derived M phi and P815 tumor target cells.
- Characterized MAF properties including solubility, heat stability, enzymatic digestion, and molecular weight.
Main Results:
- Identified MAF activity in both dense granule and cytosol fractions, with higher concentration in granules.
- Granule-derived MAF required lipopolysaccharide (LPS) for M phi tumoricidal activation.
- The MAF is a small, heat-stable peptide with a molecular weight under 10 kDa, sensitive to pepsin but not trypsin.
Conclusions:
- NK cell granules contain a potent MAF.
- This MAF is a heat-stable peptide that activates macrophage tumoricidal activity, likely playing a role in anti-tumor immunity.