Detection of function-associated molecules on rat leukemic NK cells: activation by monoclonal antibody or phorbol ester.
In the present study a rat leukemia NK cell line designated CRC- (derived from RNK-16 cells) was shown to spontaneously transform into a noncytolytic (NL) line referred to as CRC-/NL cells. CRC- and CRC-/NL cells were utilized to study pathways of NK activation by phorbol esters, calcium ionophore (A23187), and monoclonal antibody (mAb). 10(-6)-10(-7) M phorbol myristate acetate (PMA) but not phorbol didecanoate or 4-beta-phorbol activated CRC-/NL to lyse YAC-1 targets. Activated CRC-/NL cells produced 20-90% specific cytotoxicity compared to 0-5% for nonactivated cells. 10(-7) M PMA inhibited normal CRC- cytotoxicity. The optimum concentration of PMA for activation was 10(-6)-10(-7) M and 3-6 h treatment time. Augmentation of cytotoxicity by PMA occurred at different E:T ratios. The time required to reverse the PMA activation of CRC-/NL cells was approximately 9-10 h posttreatment. In an effort to attempt to differentiate pathways which initiated activation, CRC-/NL cells were treated with FAM binding mAb, or with combinations of mAb and ionophore, mAb and PMA, or PMA and A23187. mAb singly or in combination with 10(-7) M PMA increased cytotoxicity. However, A23187 either singly or when combined with PMA or mAb did not produce an augmented lysis of YAC-1 target cells. Additional experiments were conducted to determine if PMA activation was associated with FAM binding. This was accomplished by analyzing redirected killing of various FAM mAb-producing myeloma cells in the presence of 10(-7) M PMA. PMA treatment of the CRC-/NL cells caused a significant increase in the lysis of myeloma/mAb-producing cells compared to control cells. Further evidence that FAM binding was associated with cytotoxicity was presented by demonstrating specific inhibition of redirected lysis by homologous mAb. Phenotype analysis of CRC- and CRC-/NL cells demonstrated that OX-7 and OX-1 expression on CRC-/NL cells was increased by 71.8 and 86.8% respectively compared to CRC-. FAM expression (78-83% positives) by CRC- and CRC-/NL cells was not different. These experiments indicated at the functional level that rat NK cells can be activated for increased cytotoxicity by FAM-specific mAb binding and/or by treatment with the diacylglycerol analogue PMA. This implies that protein kinase C mobilization either singly or in concert with inositol-1,4,5-trisphosphate activation following FAM mAb binding may play important roles in NK cell cytotoxicity.
Evans
D L
DL
Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens.
Harris
D T
DT
Staton
D L
DL
Jaso-Friedmann
L
L
D000818:Animals; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology*; D007700:Kinetics; D007942:Leukemia, Experimental / Q000276:immunology; D051381:Rats; D015398:Signal Transduction / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000001:Calcimycin; D013755:Tetradecanoylphorbol Acetate