Mechanism of Mammalian Cell Lysis Mediated by Peptide Defensins
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Mechanism of Mammalian Cell Lysis Mediated by Peptide Defensins Evidence for an Initial Alteration of the Plasma Membrane Alan Lichtenstein Division ofHematology/Oncology, Department ofMedicine, Veterans Administration Wadsworth-UCLA Medical Center, Los Angeles, California 90073 Abstract targets, are three small (mol wt 3,900) cationic peptides termed defensins or human neutrophil peptides 1, 2, and 3 (HNP Defensins induce ion channels in model lipid bilayers and per- 1-3).' The human defensins are secreted by activated PMNs meabilize the membranes of Escherichia coli. We investigated (10) and could, therefore, function as cytotoxins during anti- whether similar membrane-active events occur during defensin- body-dependent cellular cytotoxicity. In addition, a synergistic mediated cytolysis of tumor cells. Although defensin-treated cytotoxic effect occurs when target cells are exposed to a combi- K562 targets did not release chromium-labeled cytoplasmic nation of defensins and toxic oxidants (8). It is, thus, conceiv- components for 5-6 h, they experienced a rapid collapse able that defensins may play a role in tissue injury seen during (within minutes) of the membrane potential, efflux of rubidium, inflammatory or infectious processes. and influx of trypan blue. Defensin treatment also blunted the To investigate the mechanism of defensin-induced injury, subsequent acidification response induced by nigericin, thereby we have utilized continuously cultured tumor cells as model further supporting the notion of enhanced transmembrane ion targets. In previous studies (11, 12), K562 cells exposed to de- flow during exposure. These initial effects on the plasma mem- fensins began to release radiolabeled chromium after a lag pe- brane were not sufficient for subsequent lysis; a second phase of riod of 3-4 h. Cytotoxicity depended upon energy metabolism injury was required which involved the continued presence of and cytoskeletal activities of the target ( 12). These results sug- defensin. The rapid membrane permeabilization phase was in- gested that HNP-mediated lysis was not due to a rapid creation hibited by azide/2-deoxyglucose, cytochalasin B, and increased of plasma membrane lesions and that endocytosis of HNP concentrations of extracellular potassium and was unaffected might have to occur before lysis could be realized. However, by actinomycin-D, cycloheximide, and varying the calcium con- other evidence indicates defensins rapidly damage biological centration. In contrast, the second phase was unaffected by cy- membranes. They are only active against enveloped viruses tochalasin B, inhibited by azide/2-deoxyglucose, enhanced by (13); they create ion channels in model lipid bilayers (14), and; actinomycin D and cycloheximide, and varied with calcium con- their lethal effect on Escherichia coli is due to rapid sequential centration. These results indicate the initial adverse effect of permeabilization of the outer and inner membrane (15). The defensins on mammalian cells occurs at the cell membrane. It is results of the present study indicate the initial target structure possible that the second phase of injury is mediated intracellu- for defensins on human tumor cells is also the plasma mem- larly by defensin that has been internalized through this leaky brane. However, rapid membrane permeabilization, by itself, membrane. (J. Clin. Invest. 1991. 88:93-100.) Key words: was insufficient for cell lysis. Ultimate target destruction also cytotoxicity - membrane permeabilization - neutrophils * poly- depended upon a second phase of injury which required the morphonuclear leukocyte granule proteins continued presence of HNP, thus explaining the lag in chro- mium release. The two phases of injury were independently Introduction regulated. Neutrophils (PMNs) possess at least two mechanisms by which Methods they can lyse mammalian target cells. One of these depends upon the production of toxic oxidative metabolites that are Tumor targets. The K562, Raji, L929, and YAC-l lines were main- generated from the PMNs' respiratory burst (1, 2). However, a tained in vitro by biweekly passage in RPMI media with 10% fetal calf second mechanism, often detected during studies of PMN-in- serum (FCS, Reheis, Phoenix, AZ). duced antibody-dependent cellular cytotoxicity, is indepen- Reagents. Human defensins were purified from PMNs to homoge- dent of the respiratory burst (3-5). This latter mechanism is neity as previously described (9) by applying ion exchange and reverse- less well understood, but some evidence (6, 7) suggests it is phase HPLC followed by gel exclusion chromatography on a long Bio- mediated by one or more toxic proteins released from PMN Gel P-10 column (Bio-Rad Laboratories, Richmond, CA). A mixture granules. ofthe three defensins (HNP 1, 2, and 3 in a ratio of 2:2:1 by weight) was The most potent cytolytic proteins of PMN granules, at used in all experiments except in the binding assay where iodinated least for leukemia (8) and purified HNP-1 was used (see below). (2',7')-Bis-carboxy-ethyl)-(5,6)- pulmonary (9) and endothelial (9) carboxyfluorescein acetoxymethyl-ester (BCECF-AM) was purchased from Molecular Probes, Eugene, OR. All other reagents were pur- chased from Sigma Chemical Co., St. Louis, MO. Address reprint requests to Dr. Lichtenstein, 691/Wi 1 1H, VA Wads- Rubidium release. Targets (105 in 0.1 ml) were incubated with 25 worth Hospital, Wilshire and Sawtelle Blvds., Los Angeles, CA 90073. gCi of 86RbCI in 0.1 ml of RPMI for 1 h and washed four times. Cells Receivedfor publication S December 1990 and in revisedform 15 were then incubated with or without HNP in RPMI without FCS in March 1991. The Journal of Clinical Investigation, Inc. 1. Abbreviations used in this paper: HNP, human neutrophil peptide; Volume 88, July 1991, 93-100 TPP+, tetraphenylphosphonium ion. Defensin-mediated Cell Injury 93 microtiter plates for varying durations. 4 x I04 targets were incubated blade, and the radioactivity of the pellet was assayed in a y-counter per well. Plates were then centrifuged and 0.1 ml of cell-free superna- (model I 191, Tracor, Inc., Austin, TX). The mean coefficient of varia- tant was removed and assayed in a y-counter. Percent specific release tion for counts per minute from the triplicate samples was < 5%. Con- was calculated by: (cpm,,p - cpm<OtrO.cpm.. - cpmO,4,nt) X 100. trol experiments, with ['4C]sucrose as an extracellular fluid marker, Counts per minute-maximal was determined by lysis with detergent confirmed that negligible quantities of supernatant fluid contaminated and control release was determined by incubating cells alone without the tumor cell pellet by this technique. Binding experiments were per- HNP. Maximal release was always > 90% of incorporated counts and formed in RPMI media. The trace-labeled HNP- I induced chromium control release remained < 30% of incorporated counts during the first release from K562 cells at 6 h with comparable efficiency compared to 35 min of incubation. Samples were run in quadruplicate and the stan- native preparations. The percent HNP bound was determined after dard deviation (SD) of replicates was always < 5% of the mean. incubating '25I-HNP with K562 cells for varying intervals at 370C. Chromium release assay. Targets were chromated as previously Measurement of intracellular pH. Measurements of intracellular described (7) and then washed four times. Labeled targets (104 in 0.1 ml pH (pHi) were made as previously described (I18). Briefly, the pH-sensi- of RPMI) were incubated with or without defensin (in 0.1 ml of RPMI) tive dye BCECF was incorporated into K562 cells by incubating 107 in microtiter plates at 370C for varying durations. After incubation, cells with 2 MM BCECF-AM for 30 min at 370C in a solution contain- plates were centrifuged, 0.1 ml of supernatant was removed, and ing 140 mM NaCl, 5 mM KCI, 1 mM MgCl2, 0.5 mM CaCl2, 5 mM counted in a y-counter. Percent specific chromium release was calcu- sodium pyruvate, 10 mM Hepes, 5 mM glucose, and 0.1% bovine lated as described above for specific 'Rb release. Maximal (> 90% of serum albumin (BSA). The solution was adjusted to pH 7.35-7.45 with incorporated counts) and control values (always < 25% ofincorporated saturated Tris base. Cells were then washed twice and resuspended in counts) were also determined as for ssRb release. In some experiments the same solution except that BSA was excluded. The dye signal was (see Tables II and IV), the chromium release assay was performed in 10 calibrated by releasing the dye from K562 cells with 50 MM digitonin x 75 borosilicate test tubes with 10' chromated targets incubated per and measuring the fluorescence at pH 6.1-8.2. A linear response was tube. The test tubes were centrifuged at 30 or 60 min, HNP-containing obtained in the pH range 6.3-8.0. Fluorescence was continuously re- media was removed, and targets were resuspended for continuation of corded in a fluorescence spectrophotometer (model 650-40, Perkin- the assay. Both cell-free supernatant and pellet were then counted Elmer Corp., Norwalk, CT) with excitation and emission wavelengths in a y-counter. Simple percent chromium release (in contrast to spe- of 500 and 530 nm and slits of 3 and 12 nm. Approximately 106 cells cific chromium release) was calculated as: cpms,,,icpm,.pa,, were used in each experiment. In preliminary experiments, the sponta- + cpmPu,,,) X 100. neous release of BCECF-AM from resting K562 cells incubated in the Protein synthesis. Protein synthesis was determined as previously buffer over 60 min was < 5%. described (12) by assaying [3H]leucine (New England Nuclear, Boston, Assay for trypan blue internalization. Targets (I0O in 0.1 ml) were MA) incorporation into TCA-precipitable material. After pulsing with incubated with or without defensins (in 0.1 ml) in microtiter plates. 2 MtCi/well (I0s targets per well) of [3H]leucine for 1 h, cells were precipi- Assays were run in RPMI without FCS. After varying durations of tated with 5% TCA, redissolved in 0.1 N NaOH, added to Aquasol incubation at 370C, 20 Ml of 0.4% trypan blue was added to each well (New England Nuclear), and counted in a scintillation counter.