Studies on the mechanism of natural killer cell-mediated cytotoxicity. VI. Characterization of human, rat, and murine natural killer cytotoxic factors.
Recent evidence has implicated natural killer cytotoxic factors (NKCF) as the lytic mediators of NK cell-mediated cytotoxicity reactions. The objective of this study was to examine and compare some of the biochemical and functional characteristics of human, rat, and murine NKCF. Supernatants containing NKCF were generated by stimulating effector cells with Con A or U937 (for human PBL) or YAC-1 (for rodent spleen cells) and tested for cytotoxic activity in a 20-hour (rodent) or 24-hour (human) 51Cr release assay. NKCF activity was inactivated by heating to 63 degrees C, 8 M urea, pH 2, and reduction and alkylation. These factors were highly sensitive to trypsin, moderately sensitive to papain and resistant to neuraminidase. Adsorption of human NKCF to U937 cells is inhibited by mannose-6-phosphate and adsorption of rodent NKCF to YAC-1 cells is inhibited by alpha-methyl-D-mannoside and fructose-6-phosphate. Oxidation of NKCF with sodium periodate abolished lytic activity. Pretreatment of NKCF with Con A but not pretreatment of target cells inhibited lytic activity. NKCF activity eluted in a single broad band of apparent MW of 15,000-40,000 after fractionation by HPLC gel permeating chromatography. Pooled fractions containing NKCF activity were subjected to some of the same tests performed on whole supernatants. Test result with semipurified NKCF confirmed that these factors are inactivated by trypsin or sodium periodate and that mannose-6-phosphate inhibits their binding to target cells. There were no major differences observed in NKCF produced by the three different species whether stimulated by Con A or NK-sensitive tumor cells. The evidence indicates that NKCF are glycoproteins in which disulfide bonding is essential for lytic activity. Furthermore, it appears that carbohydrate residues expressed on NKCF molecules are involved in the binding of these factors to the target cell membrane.
D000818:Animals; D002236:Carbohydrate Conformation; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D005636:Fructosephosphates / Q000494:pharmacology; D006801:Humans; D007111:Immunity, Cellular; D007694:Killer Cells, Natural / Q000276:immunology*; D055646:Killer Factors, Yeast; D008222:Lymphokines / Q000032:analysis* / Q000302:isolation & purification / Q000502:physiology; D008360:Mannosephosphates / Q000494:pharmacology; D051379:Mice; D008808:Mice, Inbred CBA; D009439:Neuraminidase / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000494:pharmacology; D010504:Periodic Acid / Q000494:pharmacology; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000502:physiology; D051381:Rats; D011916:Rats, Inbred F344; D013045:Species Specificity
D005636:Fructosephosphates; D055646:Killer Factors, Yeast; D008222:Lymphokines; D008360:Mannosephosphates; D011506:Proteins; D010504:Periodic Acid; C027693:mannose-6-phosphate; C027618:fructose-6-phosphate; C009288:metaperiodate; D009439:Neuraminidase; D010447:Peptide Hydrolases