Inhibition of human NK cell-mediated cytotoxicity by exposure to ammonium chloride.
Ammonium-chloride-containing solutions (AC) are routinely used to lyse red blood cells during preparation of PBMC. Although exposure to AC has been described to affect the ultrastructural appearance of large granular lymphocytes and to temporarily inhibit cytolytic activity of PBMC preparations, the cellular basis of this phenomenon has not been studied. Here, the inhibitory effect of AC on human CTL and NK-mediated cytotoxicity has been analyzed in 4-h 51Cr-release assays. The results show that NK killing of K562 leukemia cells and xenogeneic endothelial cells is inhibited by AC exposure. The effect is dose-dependent and reversible, because recovery of cytotoxicity is observed within 15 h of re-culturing. AC does not reduce the viability of NK cells and the inhibitory effect is not mediated by the exhaustive release of granzymes upon AC treatment. In contrast, antigen-specific CTL killing of EBV-transformed B-lymphoblastoid cell lines and xenogeneic PHA lymphoblasts was less sensitive to AC and data are presented suggesting that FasL-induced apoptosis is not inhibited by AC. In conclusion, perforin-mediated NK killing is AC-sensitive whereas CTL killing and FasL-mediated killing appear to be AC-resistant. Therefore, AC represents a powerful tool to study different mechanisms of cell-mediated cytotoxicity and may be helpful in assessing antigen-specific CTL cytotoxicity without the influence of NK cell-mediated background killing.
Brander
C
C
AIDS Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA.
Matter-Reissmann
U B
UB
Jones
N G
NG
Walker
B D
BD
Sachs
D H
DH
Seebach
J D
JD
D000643:Ammonium Chloride / Q000494:pharmacology*; D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects / Q000276:immunology; D015550:Cell Degranulation / Q000187:drug effects; D002461:Cell Line, Transformed; D003602:Cytotoxicity, Immunologic / Q000187:drug effects* / Q000276:immunology; D053222:Fas Ligand Protein; D006801:Humans; D020014:K562 Cells; D015979:Killer Cells, Lymphokine-Activated / Q000187:drug effects / Q000276:immunology; D007694:Killer Cells, Natural / Q000187:drug effects* / Q000276:immunology; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000276:immunology*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects* / Q000276:immunology
C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D000643:Ammonium Chloride; D054353:Perforin