Transmembrane chloride flux is required for target cell lysis but not for Golgi reorientation in cloned cytolytic effector cells. Golgi reorientation, N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase release, and delivery of the lethal hit are separable events in target cell lysis.
Cell-mediated cytotoxicity can be inhibited by the replacement of chloride with ions that are incapable of passing through chloride channels or by the presence of stilbene disulfonate derivatives known to interfere with chloride flux. We show that the stilbene disulfonate (4,4-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) inhibits lysis of YAC-1 targets by the cloned cell line NKB61A2. Inhibition of lysis occurs on the level of the effector cell inasmuch as preincubation of effectors but not of targets interferes with subsequent lysis. Moreover, inhibition of chloride flux in the target does not interfere with target cell lysis by cytotoxic granules isolated from killer cells. Target cell binding takes place in the presence of DIDS or absence of external chloride, suggesting that events that follow target cell binding require chloride flux. We show that reorientation of the Golgi apparatus, which occurs subsequent to target cell binding in the effector cell, occurs under conditions that interfere with chloride flux. It is therefore suggested that events in the effector cell taking place subsequent to the Golgi apparatus reorientation reaction are inhibited and that delivery of the lethal hit is a stimulus-induced secretory event that requires transmembrane chloride flux. Delivery of the lethal hit is shown to be independent of the release of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) serine esterase, suggesting that cytolytic components and BLT serine esterase are likely packaged in different vesicles.
Prochazka
G
G
Immunology Program, University of Southern California Comprehensive Cancer Center, Los Angeles 90033.
Landon
C
C
Dennert
G
G
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology / Q000502:physiology; D002470:Cell Survival; D002712:Chlorides / Q000502:physiology*; D002999:Clone Cells / Q000201:enzymology / Q000276:immunology; D003470:Culture Media; D003594:Cytoplasmic Granules / Q000502:physiology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D005942:Gluconates / Q000502:physiology; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism*; D053804:Granzymes; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors
D002712:Chlorides; D003470:Culture Media; D005942:Gluconates; D007166:Immunosuppressive Agents; D015842:Serine Proteinase Inhibitors; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D053804:Granzymes; D012697:Serine Endopeptidases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C030691:gluconic acid