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Intracellular pH and multidrug resistance regulate complement-mediated cytotoxicity of nucleated human cells
J H Weisburg1, P D Roepe, S Dzekunov
1Program in Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York 10021, USA.
Insights
Elevated intracellular pH (pHi) in multidrug resistance (MDR) cells, caused by P-glycoprotein (P-GP) or other methods, confers resistance to complement-mediated cytotoxicity (CMC). This resistance involves reduced complement deposition on the cell membrane, not altered membrane pore formation.
Area of Science:
- Cellular and Molecular Biology
- Immunology
- Biochemistry
Background:
- Multidrug resistance (MDR) cells overexpressing P-glycoprotein (P-GP) exhibit resistance to complement-mediated cytotoxicity (CMC).
- The role of elevated intracellular pH (pHi) in this resistance mechanism was previously uncharacterized.
Purpose of the Study:
- To investigate whether elevated pHi is sufficient to confer resistance to CMC in MDR cells.
- To elucidate the molecular mechanism linking P-GP, pHi, and CMC resistance.
Main Methods:
- Compared CMC in P-GP overexpressing cells and control cells with experimentally elevated pHi (CO2 conditioning, Cl- substitution).
- Utilized antibody-antigen systems, complement sources, and photometric monitoring (BCECF) to assess pHi changes and complement activity.
- Analyzed antibody binding, internalization, complement deposition, and membrane attack complex (MAC) formation.
Main Results:
- Elevation of pHi, by P-GP expression or other methods, conferred significant resistance to CMC.
- Resistance was blocked when alkalinization was inhibited, indicating pHi's crucial role.
- Reduced and delayed complement deposition on cell membranes was observed in high-pHi cells, while MAC stoichiometry remained unchanged.
Conclusions:
- Elevated intracellular pH is a key factor conferring resistance to CMC in MDR cells.
- The mechanism involves impaired complement deposition on the cell membrane, not altered MAC formation.
- Findings suggest P-GP's function extends beyond a simple 'drug pump' model, impacting cellular defense mechanisms.
Abstract:
In previous work (Weisburg, J. H., Curcio, M., Caron, P. C., Raghi, G., Mechetner, E. B., Roepe, P. D., and Scheinberg, D. A. (1996) J. Exp. Med. 183, 2699-2704), we showed that multidrug resistance (MDR) cells created by continuous selection with the vinca alkaloid vincristine (HL60 RV+) or by retroviral infection (K562/human MDR 1 cells) exhibited significant resistance to complement-mediated cytotoxicity (CMC). This resistance was due to the presence of overexpressed P-glycoprotein (P-GP). In this paper, we probe the molecular mechanism of this phenomenon. We test whether the significant elevated intracellular pH (pHi) that accompanies P-GP overexpression is sufficient to confer resistance to CMC and whether this resistance is related to effects on complement function in the cell membrane. Control HL60 cells not expressing P-GP, but comparably elevated in cytosolic pHi by two independent methods (CO2 "conditioning" or isotonic Cl- substitution), are tested for CMC using two different antibody-antigen systems (human IgG and murine IgM; protein and carbohydrate) and two complement sources (rabbit and human). Elevation of pHi by either of these methods or by expression of P-GP confers resistance to CMC. Resistance is not observed when the alkalinization mediated by reverse Cl-/HCO3- exchange upon Cl- substitution is blocked by treatment with dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate. Continuous photometric monitoring of 2',7'-bis(carboxyethyl)-5, 6-carboxyfluorescein (BCECF), to assess changes in pHi or efflux of the probe through MAC pores, in single cells or cell populations, respectively, verifies changes in pHi upon CO2 conditioning and Cl- substitution and release of BCECF upon formation of MAC pores. Antibody binding and internalization kinetics are similar in both the parental and resistant cell lines as measured by radioimmunoassay, but flow cytometric data showed that net complement deposition in the cell membrane is both delayed and reduced in magnitude in the MDR cells and in the cells with increased pHi. This interpretation is supported by comparison of BCECF release data for the different cells. Dual isotopic labeling of key complement components shows no significant change in molecular stoichiometry of the MACs formed at different pHi. The results are relevant to understanding clinical implications of MDR, the physiology of P-GP, and the biochemistry of the complement cascade and further suggest that the "drug pump" model of P-GP action cannot account for all of its effects.