Comparative studies of the cytotoxic T lymphocyte-mediated cytotoxicity and of extracellular ATP-induced cell lysis. Different requirements in extracellular Mg2+ and pH.
We recently proposed that extracellular ATP (ATPo) may be involved in CTL-mediated cytotoxicity by acting in concert with yet unidentified cellular components (ATPo receptors/ATPo-binding proteins, ectoprotein kinases). The TCR-triggered ATPo accumulation by CTL has been demonstrated, whereas the resistance of CTL to ATPo was explained by the action of highly active ecto-ATPases or by the absence of relevant ATP-binding proteins. However, no data were available to discriminate between the possibilities of: i) ATPo acting alone as a "hit" molecule because of the cell-permeabilizing properties of ATP4- or ii) ATPo acting as a "messenger" (as MgATP2-) in concert with other molecules. Comparing ATPo-induced and CTL-mediated cell lysis, we found that ATPo-induced lysis of some target cells is greatly decreased at neutral and acidic pH, whereas Ca(2+)-dependent CTL-mediated lysis of the same cells is barely affected. In agreement with the observed pH dependency, at low Mg2+ concentrations, which favor ATP4- over MgATP2-, maximal ATPo-induced lysis was observed. However, CTL-mediated cytotoxicity in both Ag-specific and retargeting assays was markedly reduced at low Mg2+ concentrations. These results suggest that ATPo acting alone as a "hit" molecule cannot fully account for the extracellular Ca(2+)-dependent lethal hit delivery by CTL or that ATP4- is active at very low concentrations. This conclusion was further supported by studying the lytic effect of ATPo and CTL on the anti-TCR mAb-coupled SRBC. CTL were efficient in the SRBC lysis, whereas no lysis of SRBC by ATPo was detected. The resistance of SRBC to ATPo is not caused by a high ATPo degradation, because the ecto-ATPase activity of SRBC was much lower than in ATPo-resistant CTL OE4 cells and comparable with EL4 tumor cells, which were easily lysed by ATPo. These data suggested the need for careful consideration of the pH and cation composition of the media used for studying ATPo effects. The caveats in the use of ATP-degrading enzymes to implicate the role of extracellular ATPo in the CTL-mediated cytotoxicity are described here. A clarification of the previously described cytotoxicity inhibition by hexokinase, which is caused by an inhibitory salt effect, is presented. It is suggested that if Ca(2+)-dependent lysis of SRBC and of other target cells by CTL does involve extracellular ATP, it may function as a "messenger" in concert with other extracellular molecules.(ABSTRACT TRUNCATED AT 400 WORDS)
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
F
F
Redegeld
A
A
Filippini
M
M
Sitkovsky
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000502:physiology; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002463:Cell Membrane Permeability; D003602:Cytotoxicity, Immunologic; D006863:Hydrogen-Ion Concentration; D007111:Immunity, Cellular; D066298:In Vitro Techniques; D008274:Magnesium / Q000502:physiology; D051379:Mice; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium