Synergistic cytolysis mediated by hydrogen peroxide combined with peptide defensins

CELLULARIMMUNOLOGY

Synergistic

114,104-l

16 (1988)

Cytolysis Mediated by Hydrogen with Peptide Defensins

Peroxide Combined

ALAN K. LICHTENSTEIN,**~TOMAS GANZ,-~§ MICHAEL E. SELSTED,@~.~ AND ROBERT I. LEHRER*+~I *Department ofkfedicine, V. A. Wadsworth-UCLA Medical Center, Departments of TMedicine and *Pathology, UCLA Center for the Health Sciences, 4 Will Rogers Pulmonary Research Laboratory, and llthe Jonsson Comprehensive Cancer Center, Los Angeles, California 90073 Received November 3,1987; accepted January 9,1988 Possible cytolytic interactions between hydrogen peroxide (H202) and neutrophil granule proteins were studied. Preliminary experiments demonstrated synergistic cytolysis when erythroleukemia targets were exposed to H202 combined with a low molecular weight (-3900) granule extract that was predominantly composed of peptide defensins. The synergistic interaction was confirmed when sublytic concentrations of Hz02 were combined with defensin preparations that had been purified to homogeneity. Synergy was concentration dependent in regard to both molecules and could not be explained by trace contamination of defensin preparations with myeloperoxidase. Sequential addition experiments suggested that synergistic lysis required a simultaneous exposure to both cytotoxins. In the presence of sublytic concentrations of H202, the binding of iodinated defensin to targets was significantly increased, providing a possible explanation for the observed synergy. Since both molecules are concurrently secreted by activated neutrophils, this interaction may be important during leukocyte-mediated anti-tumor eff%tS or hflaffUXit0~ tk.We injury. 0 1988 Academic PITS, Inc.

INTRODUCTION Stimulated neutrophils (PMNs) possess two separate mechanisms by which they can induce cellular injury. One of these depends upon the production of reactive oxygen intermediates (ROIs) generated from reactions of the oxidative burst. Hydrogen peroxide (HzOz), a major product of the burst, can lyse murine tumor cells (I), some human tumor cell lines (2,3), and several types of nonmalignant cellular targets (4-6). The second potential mechanism of lysis is nonoxidative and is mediated by one or more protein cytotoxins contained within the PMN’s lysosomal compartment. Elastase (7), cathepsin-G (g), and defensins (9), all located in primary azurophilit granules, have been demonstrated to lyse a variety of human or murine target cell types. The physiologic role of these two mechanisms acting independently in humans is not entirely clear for several reasons: First, most human tumor cells, fibroblasts, erythrocytes, and lymphocytes appear markedly resistant to the lytic effects of H202 (10). In other human targets, effective lysis by Hz02 is seen only when concurrent inhibition of the glutathione redox cycle is achieved (5,10). Second, most PMN stimuli selectively induce external release of secondary granule constituents (11) and the 104 OOOS-8749188 $3.00 Copyri&t 0 I988 by Academic F‘res, Inc. AU tights of reproduction in any form reserved.

DEFENSIN-H202

SYNERGISTIC

LYSIS

105

extracellular concentrations of the cytotoxic primary granule proteins may not be sufficient to independently lyse adjacent cell targets. Since there are these questions concerning the independent roles of H202 and granule proteins, we investigated a possible interaction between these two cytotoxins during target cell lysis. Preliminary experiments indicated synergistic cytolysis could occur when targets were exposed to the combination of H202 and granule extracts containing defensins. Theoretically, both would be simultaneously secreted by activated PMNs into the vicinity of adjacent targets. This interaction was then confirmed by using H202 in combination with purified defensins and some of its characteristics were defined. MATERIALS

AND METHODS

Tumor lines. Human (K562, Raji, PA-l, SK-OV3) and murine (YAC-1) tumor lines were maintained in RPM1 medium with 10% fetal calf serum (FCS, Reheis, Phoenix, AZ). Erythrocyte (RBC) targets. Human heparinized blood was washed in phosphatebuffered saline (PBS) three times with removal of plasma and bully coat. After suspension in phosphate-buffered Hanks balanced salt solution RBCs were chromated as described below. Chromium release assay. Tumor or erythrocyte targets were incubated with 50 PCi of ‘ICr for 1 hr at 37°C in RPMI-10% FCS. Labeled targets ( lo4 in 0.1 ml of RPMI) and varying concentrations of PMN granule extracts, reagent grade H202, purified human defensins, or combinations of these agents (in 0.1 ml of RPMI) were incubated in microtiter plates at 37°C in 5% COz. All assays were run in the absence of FCS since serum has inhibitory effects on the lytic activities of defensins and H202. After varying durations, the plates were centrifuged ( 1200 rpm X 10 mins) and 0.1 ml of supematant was counted in a gamma counter. All samples were run in quadruplicate. Percentage lysis was calculated as (cpm,,, - cpmcont&/(cpmmaximd - cp&,& X 100. Control release was determined from targets incubated in media alone (less than 25% incorporated counts) and maximal release was achieved by adding 1 A4 HCl (greater than 90% incorporated counts). The standard deviation of quadruplicate samples was always less than 5% of the mean. The concentration of Hz02 causing 50% specific lysis (LD50) was calculated by interpolation as previously described ( 1). In similar fashion, the concentration of defensin causing 30% specific lysis (LD& was also calculated. Preparation of PMN granules and granule protein extraction. PMNs were suspended in 50 ml of 0.34 A4 sucrose (adjusted to pH 7.4), homogenized in a PotterElvehjem homogenizer (four cycles at 2-2.5 min each cycle) and centrifuged at low speeds (200g for 10 min) to remove nucleii and cellular debris. The granule-containing supematants were pooled and the granules were sedimented at 27,000g for 20 mins. Granule protein extraction was performed by the method of Modrzakowski et al. (12) as modified by Greenwald and Ganz (13). Briefly, the granule sediment was extracted three times in 60 ml of 0.2 M sodium acetate buffer with 0.01 M CaC12 (pH 4). Each extraction was done overnight at 4°C and the residue was separated by centrifugation at 27,000g for 20 min. The extracts were concentrated by ultrafiltration and placed on a Sephadex G- 100 column. Approximately 44 mg of extract was eluted with 0.2 M sodium acetate buffer (pH 4) and collected in lo-ml fractions. The

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ET AL.

VOLUME

FIG. 1. Preparative chromatogram of crude PMN granule extract fractionated on a Sephadex G-100 column. Arrowheads show where fractions were separated.

fractions were pooled into four peaks (A, B, C, and D) and three valleys (AB, BC, CD) as defined by their A 280 pattern, concentrated by ultrafiltration, and dialyzed against PBS with Spectrapore 6 tubing, MW 3000 cutoff. A total of 39 mg of protein was recovered and was distributed as follows: Peak A, 13.7%; Peak B, 22.5%; Peak C, 14.2%; Peak D, 29.7%; Valley AB, 7.2%; Valley BC, 4.9%; Valley CD, 8%. These recoveries are similar to those achieved by Modrzakowski et al. ( 12, 14). The extract fractions were stored at 4°C and tested within 2 weeks oftheir generation. The preparative chromatogram of the crude PMN granule extract fractionated on a Sephadex G- 100 column is shown in Fig. 1. Purification ofdefensins. The human defensins HNP- 1, HNP-2, and HNP-3 were obtained from normal PMNs and purified to homogeneity as previously described (15) by applying sequential ion-exchange and reverse-phase HPLC followed by gel exclusion chromatography on a long Bio-Gel P-10 column. Lyophilized defensins were dissolved in 0.0 1% acetic acid at 2 mg/ml and stored at -20°C until use. Purity was confirmed by demonstrating single bands on acid urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide concentrations were determined by amino acid analysis. The combination of HNP- 1:HNP-2:HNP-3 mixed in a 2:2: 1 ratio by weight was used in chromium release assays. Purified HNP-1 was used in binding studies. As previously described (9), the degree of tumor lysis was comparable between these two reagents. Iodination of HNP-I. Two milligrams of lyophilized HNP-1 was dissolved in 0.5 ml of 0.05 M sodium phosphate (pH 7) and 2 mCi of Na’*‘I ( 1.07 mmol) was added in a volume of 20 ~1 of 0.1 N NaOH. Two Iodo-Beads (Pierce Chemical Co., Rockford, IL) were immediately added, and the mixture was incubated for 45 min at room temperature with occasional stirring. Cold KI was added in fivefold (molar) excess with respect to peptide. After a lo-min incubation at room temperature, the iodinated peptide was desalted on a 1.O by 25-cm Cq reversed-phase column (Vydac, The Separations Group, Hesperia, CA). Iodinated peptide was eluted at 1.5 ml/min by using as solvents 0.1% trifluoroacetic acid (TFA) in water (solvent A) and 0.1% TFA in acetonitrile (solvent B). The iodination mixture was injected onto the column, and the elution gradient was developed linearly from 0 to 60% B over 60 min. The iodinated peptide was lyophilized and quantitated by amino acid analysis. Iodinated and native preparations of HNP- 1 killed K562 targets with equal efficiency. Binding assay. The assay was a modification of the previously described ( 16) system which assessed binding of rabbit defensin to Candida. Briefly, 5 X lo5 viable K562 cells in a volume of 1 ml were exposed to HNP-1 that had been trace labeled with ‘25I as described above. Incubations were performed in triplicate in 1.5-ml conical polypropylene centrifuge tubes (West Coast Scientific, Berkeley, CA) that contained 0.4 ml of silicone oil (Versilube F50; General Electric Co., Waterford, NY).

DEFENSIN-H202

SYNERGISTIC

LYSIS

107

After incubation at 37°C for varying intervals, the tubes were centrifuged for 90 set at 12,000g in an Eppendorf Model 3200 microcentrifuge (Brinkmann Instruments, Inc., Westsbury, NY). Separation of cells from supematant was complete within 30 sec. After the supematant and oil layers were removed, the tip of the tube was amputated with a razor blade, and the radioactivity of the pellet was assayed in a Tracer Model 119 1 gamma counter. The mean coefficient of variation for cpm for the triplicate samples was less than 5%. Control experiments, using [‘4C]sucrose as an extracellular fluid marker, confirmed that negligible quantities of supematant fluid contaminated the tumor cell pellet by this technique. Binding experiments were performed in RPM1 media. Statistics.The Student t test was used to test for significance of differences. Reagents. Catalase and sodium azide were obtained from Sigma Chemical Co. (St. Louis, MO). Myeloperoxidase was a generous gift from Dr. Ingel Olsson. It was isolated from leukocytes obtained from a patient with chronic myelogenous leukemia as previously described (17). MPO was assayed by the U-dianisidine technique. RESULTS

Preliminary Experiments: Synergistic Lysis from Combined Treatment with Hz02 and PAIN Granule Extracts PMN granules were extracted and the extracts fractionated by size as previously described ( 13). Two of the four protein peaks, peaks B (MW = 30,000) and D (MW - 3900) exerted significant lysis of K562 targets in a 6-hr chromium release assay. At 50 pg/ml, specific lysis achieved by peak D ranged from 16 to 24% and that by peak B ranged from 13 to 18%. Peaks A and C were totally ineffective as lytic molecules while valleys AB, BC, and CD resulted in minimal lysis which was probably due to small amounts of the two lytic protein peaks present in the valleys. Peak B contains elastase, cathepsin-G, and other proteases (13) while peak D almost entirely (>95%) consists of the three defensins, HNP-1, -2, and -3 (13). Both peaks were combined with sublytic concentrations of Hz02 to test a possible interaction. When 10e4 M Hz02 (sublytic by itself) was combined with 50 gg/ml of peak D, lysis increased from 2 1 to 32% (mean of three experiments). Likewise, when 5 X 1O-4 M H202 (also sublytic) was combined with 20 or 50 gg/ml of peak D, lysis increased from 5 to 28% and from 21 to 49%, respectively. Combinations of low5 M H202 with 5,20, and 50 pg/ml of peak D yielded only additive results. In no instance was there any synergy when peak B was combined with varying sublytic concentrations of H202. Because of these preliminary results, we further investigated interactions between H202 and purified defensins.

Dose-Response of Tumor Lysis by H202 and HNP-I-HNP-3 The tumor-lytic effects of H202 and purified HNP- 1-HNP-3 were first determined in detail on an individual basis. As shown in Fig. 2, K562 targets are quite resistant to Hz02 with multiple experiments demonstrating an LDSo of 8 X 1O-3 A4. This resistance is not peculiar to K562 targets, as experiments with other human tumor lines (Raji, PA- 1, and SKOV3) have demonstrated a comparable LDso for H202 lysis (not shown). In contrast, murine targets are considerably more sensitive to H202 as demonstrated by lysis of murine lymphoma YAC- 1 targets (Fig. 2, LDso = 3.5 X 10s5 M) and MOT targets (9.5 X 10e6 M; (18)). In our experience, H202 lysis always exhibits

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100 1

[H,O,l M RG. 2. Lysis of KS62 (0) or YAC-1 (A) targets by varying concentrations of H202. Several groups were also assayed in the presence of catalase, 5000 U/ml (X). Data are means f SD of five experiments.

a steep dose-response curve where targets shift from almost complete resistance to maximal lysis within 1 log molar concentration of Hz02 as shown in Fig. 2. Also, Fig. 2 demonstrates that HzOz-mediated lysis was completely abrogated in the presence of 5000 U/ml of catalase. These LDSo values for human and murine targets are comparable to those of other studies (1, 10). Figure 3 demonstrates the concentration dependence of K562 tumor lysis mediated by purified HNP- I-HNP-3. Lysis tends to plateau between 40 and 100 pg/ml. As shown, cytotoxicity is also time dependent and as low as 1 &ml HNP- I-HNP-3 exerts significant cytolysis when the assay duration is 10 hr. For the purpose of this study where cytotoxicity was usually assayed at 6 hr, 20 pg/ml was sufficient for significant albeit modest lysis while 10 cLg/ml was usually ineffective. The concentration resulting in 50% lysis was approximately 60 &ml of HNP-l-HNP-3.

100

1

FIG. 3. Lysis of K562 targets by varying concentrations of HNP-l-HNP-3. Different curves represent results from assaysperformed at different time durations (3-10 hr, shown to right of graph). Data are presented as means + SD of three experiments.

DEPENSIN-H202

SYNERGISTIC

LYSIS

109

H202 FIG. 4. Effect of HNP-I-HNP-3 on Hz02 lysis of K562 targets. Top portion shows lysis by HNP used alone (left of curves, mean + SD, four experiments, for HNP concentrations of 5,20, and 40 &ml), or in combination with varying concentrations of H202. In bottom portion, lysis due to HNP alone was subtracted from all points on the combination-treatment curves. The LDSo is shown to the right of the curves for H202 combined with 0,5,20, or 40 &ml HNP- 1-HNP-3. Data are presented as means + SD of four experiments.

Synergistic Lysisfrom Combined Treatment with H202 and Purified HNP-l-HNP-3 Synergistic lysis was observed between several concentrations of H202 and purified HNP-I-HNP-3. For clarity, the data are presented in two figures (Figs. 4 and 5). Figure 4 demonstrates the effect of varying concentrations of HNP on HzOz lysis of K562 targets. The top portion demonstrates the actual lysis by HNP alone (at $20, or 40 &ml), by Hz02 alone (o), and by all combination of the two cytolysins. In the bottom portion, the lytic response mediated by HNP alone was subtracted from all combination responses so that the effect on LDsO for H202 lysis could be isolated. The H202 dose-response curve is shifted to the left for all HNP concentrations and the degree of synergy was strongly correlated with the amount of HNP tested (Fig. 4, bottom). The LDsO’s (for Hz02 lysis) in the presence of 5, 20, and 40 pg/ml HNP (shown to the right of the figure) were 2.6, 13-, and 40-fold decreased, respectively. Synergistically enhanced lysis was primarily present between 1O-5 and 1Oe3 M H202. The maximal lytic response from H202 at 1O-2 A4 was not altered by the presence of any concentrations of HNP. Figure 5 demonstrates the effect of varying concentrations of H202 on lysis by HNP. The top portion presents the actual data and, in the bottom portion, the lytic response mediated by H202 alone was subtracted from combination responses. As shown, synergistic responses were present for concentrations of H202 between 10e5 and 1Oe3M. Not shown are the curves for HNP lysis in the presence of 1OW6and 1Om7 M H202 which were identical to that when H202 is not present. HNP lysis in the presence of lop2 M H202 is 0 (Fig. 5, bottom) since cytotoxicity mediated by that concentration of H202 used alone (75-85%) is not increased when any concentration of HNP is present. Figure 5 shows that synergy also correlates with the H202 concen-

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ET AL.

FIG. 5. Effect of Hz02 on HNP lysis of targets. Top portion shows lysis by varying concentrations of HNP (O-40 pg/ml) in combination with several different concentrations of H202 (shown to right of the curves). In the bottom portion, lysis due to Hz02 alone was subtracted from all points on the combinationtreatment curves. The LDu, is shown to the right of the curves for HNP combined with 0, 10b5, 10S4, 5 X 10e4, 1O-’ and 1O-* M H202. Data are presented as means f SD of three experiments.

tration tested. The LD~o of HNP lysis (shown to the right of Fig. 5) decreased as the sublytic concentration of Hz02 increased between 1Om5and 1Oe3M. With all synergistic combinations of HNP and H202, catalase (5-10,000 U/ml) was capable of preventing tumor lysis (not shown). In contrast, the presence of sodium azide (1 rnJ4) did not prevent synergistic lysis (not shown), suggesting that enhanced lysis was not due to an interaction between Hz02 and any myeloperoxidase which could have contaminated the HNP during its preparation from PMN granules. The phenomenon of synergy is obvious primarily because it occurred even when sublytic concentrations of Hz02 were used. For example, combining 5 X 10e4 M Hz02 (achieving 0% lysis by itself) with 20 &ml of HNP (22% lysis by itself) resulted in 50% specific lysis, 28% beyond the expected sum. In likewise fashion, combining 10e5 M H202 (ineffective by itself) with 20 pg/ml of HNP (22% lysis by itself) resulted in 35% lysis, 13% above the expected sum. To confirm the synergistic interaction algebraically, the following formula was used ( 19): AC/Ae+ SC/B,,where A, and B, represent the concentrations of H202 and HNP used in the combination to give 50% lysis (5 X 10e4 M and 20 Ilg/ml) and A, and B, represent the concentrations of the agents giving 50% lysis when used alone (8 X 10e3 M and 60 kg/ml, respectively). If AC/A, + BJB, < 1 (it actually = l/ 16 + l/3), a synergistic interaction is confirmed.

Kinetics of Synergistic Lysis As described in Fig. 3, HNP-mediated lysis is time dependent with very little chromium release detected at 3 hr. To test whether synergy with H202 includes an acceleration ofthese kinetics, we assayed lysis after varying time intervals (Fig. 6). As shown,

DEFENSIN-Hz02

SYNERGISTIC

111

LYSIS

H202(10-2M) f HNP (40p/ml)

ALONE H202(6x10-4~)

75-

v, v) 3

HNP (20pglml

50-

HNP ALONE

H202(Sx~O-4~) (40j@“l)

*

25-

TIME OF ASSAY (HRS) FIG. 6. Kinetics of K562 lysis. Chromium release assaysperformed at 1,2,3, or 6 hr for targets incubated with H202 alone (5 X 10F4, 10m2M), HNP alone (20, 40 &ml), or two combinations (20 &ml HNP + 5 X 10v4 M H202; 40 &ml HNP + 5 X low4 M H202). Data are presented as means k SD of four experiments.

chromium release is not detected at 3 hr when K562 targets are incubated with 20 or 40 pg/ml of HNP although, by 6 hr, significant lysis is present. When 20 pg/ml of HNP is combined with a sublytic concentration of Hz02 (5 X 10e4 M), synergistic lysis is observed at 6 hr but not 3 hr. However, in the presence of 40 pg/ml HNP and H202, significant lysis is detected at 3 hr. Even though present at 3 hr, synergistic lysis (between 40 pg/ml HNP and HzOz) at 6 hr is much more evident. As also shown in Fig. 6, lysis mediated by IO-’ MHz02 is much more rapid than either HNP-mediated or synergistic lysis, becoming maximal by 2 hr. Thus, a slight acceleration in kinetics is present when 40 pg/ml HNP was combined with 5 X 10V4 M H202. However, in general, the kinetics of synergistic lysis are more akin to that of HNP used alone rather than lytic concentrations of H202. Synergistic Lysis of K562 Is Not Enhanced by Myeloperoxidase

(MPO)

To further investigate possible interactions between defensins and products of the respiratory burst, MPO (26 mu/ml) was added to synergistic combinations of H202 and HNP. The chloride concentration during these experiments was 100 n&f. Preliminary experiments indicated no increase in H20z-mediated lysis of K562 cells when this concentration of MPO was added, although MPO was able to enhance H202-mediated lysis of RBCs (see below). As shown in Table 1, although 40 pg/ml HNP combined with 10-j MHz02 resulted in synergistic lysis (line H; 62% compared to the expected sum of 36%), the addition of MPO had no further enhancing effect (compare line H to line K). Furthermore, the inability of MPO to increase the H202mediated lysis of K562 targets further supports the notion that synergy between HNP and H202 can not be due to trace contamination of defensins with MPO. Lack of Synergy against Erythrocyte Targets Since K562 targets are relatively sensitive to HNP and resistant to H202, we investigated whether erythrocyte targets, which are more sensitive to Hz02 and resistant

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TABLE 1 Lack of Enhancing Effect of Myeloperoxidase on Synergistic Lysis” HNPb (A) (B) (a 03 (El F) 63 (HI (1)

+ + + + +

(J)

+

(IO

+

(M)

H202

-’ lo10-4 10-r 10-5 10-4 10-3 10-r 10-4 10-3

MPO’

Percentage lysisd

Expected sum

Increase

+ + + +

36.2 + 3 0 3 kO.2 0 0 43.9 f 3. I’ 49 k2.8’ 62.1 + 5’ 45.8 -t 4.4 47.9 + 5.2 64.4 k 4.1

36.2% 39.2% 36.2% 36.2% 39.2% 36.2%

1.1% 9.8% 25.9% 9.6% 8.7% 28.2%

’ Six-hour chromium release assay with KS62 targets incubated with HNP, H202, MPO, or various combinations. b HNP- I-HNP-3,40 pg/ml. ’ Myeloperoxidase (MPO) = 26 mu/ml. ‘Mean + SD of three separate experiments. e Lysis significantly (P < 0.05) greater than that of group incubated with defensin alone (group A).

to HNP, could be lysed in synergistic fashion by combinations of the two cytotoxins. As shown in Table 2, RBC targets are sensitive to lysis by 10B4 and 1Om3A4 H202 in contrast to resistant K562 targets. A full Hz02 dose response for RBCs (not shown) gave an LDSo of 8.5 X 10e4 M. On the other hand, RBCs are considerabIy resistant to HNP with only 5.1% lysis in the presence of 50 Kg/ml. Although Hz02 and HNP interacted to achieve synergistic lysis of K562 targets, the resulting cytotoxicity of

TABLE 2 Lack of Synergy with Erythrocyte Targets” Target RBC

K562 HNPb (A) 09 (Cl W 03 03 63

+ + + +

Hz02

(M)

10-5 10-4 10-r 10-S 1o-4 10-r

Percentage lysis’

Expected

Percentage lysis’

Expected

3823 0 0 4+-l 45 -I- 3.5d 53 k 3. Id 11 +-4.2d

38% 38% 42%

5.1 f I 4.5 f 0.8 23.4 f 1.9 65.0 + 3.1 7 +I 26 f 3.5 62 24.4

9.6% 28.5% 70.1%

‘Chromated K562 or erythrocyte targets incubated with HNP, H202 or combinations in a 6-hr chromium release assay. b HNP- 1-HNP-3,50 &ml. ‘Mean k SD ofthree separate experiments. d Lysis significantly (P < 0.05) greater than that of group incubated with defensin alone (group A).

DEFENSIN-H202

SYNERGISTIC

113

LYSIS

TABLE 3 Effect of Sequential Addition of Cytotoxins to Targets’ Compound present Preincubaton (1 hr)

(A) 09

Hz02=

HNPd (C) HNP + Hz02 0) H202 HNP (E)

In chromium release assay(6 hr) Hz02

HNP HNP + H202 HNP Hz02

Percentage lysis b -t SD

0% 14+2 42 + 5’ 12+3 16+3

’ Chromated K562 targets incubated with Hr02, defensin, or both for 1 hr, washed 1X, and then incubated for 6 hr with H202, HNP or both in combination. Percentage lysis determined after the 6-hr incubation. ’ Percentage lysis, mean + SD of three experiments. c H202 at 5 X 10m4M. d HNP at 25 rglml. e Lysis significantly (P < 0.05) greater than that of group incubated with HNP alone for 6 hr (group B or D).

RBCs was almost identical to the expected sum of their individual effects (Table 2). In other experiments not shown, the addition of MPO (26 mu/ml) to H202 resulted in enhanced lysis of RBC targets (decreasing the LD=,t, from 8.5 X 10m4 to 9 X lop5 M). However, lysis could not be further enhanced by the addition of HNP.

Characteristics of Synergy To determine whether synergy requires the simultaneous presence of both agonists, a sequential addition study was performed. Chromated K562 targets were first exposed to a sublytic concentration of Hz02 or HNP for 1 hr. The cells were then washed and resuspended in medium containing HNP, H202, or both (synergistic combination) for a 6-hr chromium release assay. The viability of targets after the 1-hr preincubation assessed by trypan blue exclusion was equal to a control group preincubated in medium alone (>94% viable). As shown in Table 3, neither of the sequential additions resulted in synergistic lysis. In addition, the pretreatment exposures to either Hz02 or HNP did not prevent the subsequent synergy resulting from a 6-hr incubation with the two toxins combined. Several other dose combinations were assayed in sequential addition experiments ( lop4 and 10e5 A4 Hz02 with 5, 10, and 15 pg/ml HNP) and in no case was there evidence of synergistic lysis. The data suggest that synergistic lysis requires a simultaneous exposure to the two cytotoxins.

Efect of Sublytic Concentrations of Hz02 on Binding of HNP to Targets One potential mechanism by which synergistic lysis is achieved could be an enhancement of HNP binding to targets in the presence of H202. Previous work (20) has demonstrated that ‘251-labeled defensin (HNP- 1) binds extensively to K562 targets with biphasic kinetics. Almost 20% of added defensin bound to K562 targets within 5 min. A secondary phase of progressive binding began at approximately 10 min and gradually increased until 60 min at which time it plateaued with overall binding of approximately 35-60% of added HNP-1. In addition, subsequent death of

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TABLE 4 Increased Binding of HNP to Targets in the Presence of Sublytic Concentrations of H202’ Percentage bound b 5 min

03202)

Expt. No. 1 Expt. No. 2 Expt. No. 3

Expt. No. 4

0 5 x 10-4M 0 10-j M 0 5 x 10-4M 5 X 10e4Mpre-Rxd 0 5 x 10-4M 5 X 10e4 Mpre-Rxd

29 f 1.7 28 +2.1 17 k2.6 13 k2.0 19.7 + 3.3 17.8 + 3.8 20.0 + 3. I 21 22.2 32 ? 1.9 31 + 1.8

60 min 34.6 46.0 31 43 29 40 32 44.9 59.3 46.2

f 2.5 k 3.2’ k4.2 -+4.5c f 3.8 k2.1C k2.4 + 4.0 f 3.6’ + 3.7

’ Binding of iodinated HNP-1 (20 &ml) to K562 targets in the presence or absence of H202 after 5 or 60 min of incubation at 37’C. b Data presented as percentage binding of added cpm, mean f SD of triplicate samples. ‘Binding significantly (P < 0.05) greater than that of corresponding group incubated in the absence of H202.

d Incubation of K562 in designated concentrations of H202 followed by washing 1X and incubation with iodinated HNP- 1 for 5 or 60 min.

targets depended upon membrane binding of HNP since conditions that impaired binding, namely low temperature, the presence of fetal calf serum and heparin, effectively prevented tumor cytolysis. Table 4 demonstrates that in the presence of sublytic concentrations of H202, the binding of HNP to targets is significantly increased when assayed at 60 min although the values after 5 min of binding were not different. The mean binding (of the four experiments) at 60 min increased from 34.9 to 47% in the presence of H202. From the molecular weight of HNP- 1 (3900), we have calculated that exposure to these concentrations of Hz02 allows an increase in binding from 2.1 X 10’ to 2.83 X 10’ molecules of HNP- l/target. The data presented in Table 4 also suggest that the enhancement of binding requires the simultaneous presence of both molecules. When targets were first incubated for 1 hr with 5 X low4 M H202, washed, and then assayed for binding of iodinated defensins (Table 4, Experiments 3 and 4), no increase could be detected. DISCUSSION The results of this study confirm a synergistic interaction between H202 and peptide defensins in target cell cytolysis. The degree of synergy was concentration dependent in regard to both cytotoxins: As the concentration of defensin rose between 5 and 50 pg/ml and the concentration of H202 increased between lop5 and lop3 M, synergistic enhancement of lysis increased. Synergy was not due to a trace contamination of defensin preparations with myeloperoxidase because (i) The presence of azide did not prevent synergy, and (ii) Partially purified MPO itself could not synergistically interact with HzOz to increase K562 lysis. Previous studies have also indicated that leukocyte-generated ROIs can interact synergistically with products secreted by nonoxidative mechanisms. Adams et al. (2 1)

DEFENSIN-H202

SYNERGISTIC

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115

described synergistic lysis of tumor targets by combined treatment with the macrophage products H202 and cytolytic factor, a neutral serine esterase. In other studies analogous to ours, Yazdanbaksh (22) detailed synergistic extracellular killing of Schistosoma mansonii by eosinophil or PMN cytoplasts and eosinophilic cationic protein (ECP). Cytoplasts obtained from a patient with CGD were ineffective, confirming that the interaction involved production of ROIs by cytoplasts. Since these cytoplasts are devoid of peroxidase, it is very likely that H202 is the moiety interacting with ECP. Thus, Hz02 can synergistically combine with lysosome-based cationic peptides for helminthotoxic as well as cytotoxic effects. It is unknown whether synergy is due to facilitating effects of Hz02 on HNP-mediated lysis or vice versa. The sequential addition experiments did not help in this regard and only indicate that a simultaneous exposure to both molecules is required. The kinetics curves (Fig. 6) suggest that synergistic lysis is more consistent with lysis due to enhanced lytic effects of HNP rather than lytic effects of H202. However, these curves compared lysis by HNP to that of lytic concentrations of H202 (lo-’ M) and they may not be relevant to lysis by HNP combined with sublytic concentrations. There are several possible explanations for the mechanism of synergistic lysis. One possibility is that sublytic concentrations of Hz02 perturb the target membrane which facilitates entry of HNP into the cell. In support of this hypothesis, Table 4 demonstrates increased target binding and/or uptake of defensin molecules in the presence of H202. The requirement for simultaneous exposure to H202 in order to achieve increased HNP binding correlates with the requirements for enhanced lysis (Table 3) and suggests that the effect of Hz02 is on defensin molecules rather than on target cell membranes. An alternative explanation is that either of the toxins could inactivate tumor cell defenses against the second. For example, sublytic concentrations of H202 cause a transient activation of poly(ADP)-ribose polymerase resulting in a reversible loss of NAD and ATP and subsequent inhibition of protein synthesis in treated targets (23). Exposure of K562 targets to the protein synthesis inhibitor cycloheximide markedly enhances lysis by defensins, suggesting that targets can repair sublethal injury mediated by HNP (unpublished data). It is, thus, possible that sublytic concentrations of H202 sensitize targets to HNP lysis by inhibiting protein synthesis. Since these H202mediated effects on protein synthesis are reversible when sublytic concentrations are used (23), this could also explain the requirement for simultaneous exposure to achieve synergy. Of interest, erythrocytes do not contain poly(ADP)-ribose polymerase (24) and we could not demonstrate synergistic lysis with these targets. According to published data, the theoretical extracellular concentration of H202 and defensins achieved by activated PMNs are sufficient for synergistic cytotoxic interactions to occur. There is approximately 6 X 10m6pg of HNP/PMN of which 5% is released following stimulation (25). In a hypothetical situation where 2 X 10’ PMNs exist in 1 ml of fluid, a potential concentration of 6 pg/ml of HNP can be reached. According to data from Test and Weiss (26), the same activated PMNs could secrete H202 to reach an extracellular concentration of 8 X 1Om4M. A combination of H202 and HNP in these concentrations could result in slightly greater than 25% target lysis. Furthermore, the extracellular concentrations of H202 and defensin are theoretically increased more than lOOO-fold if secretion is directed entirely into a PMN-target cell cleft containing a volume of only l-5 pm. In summary, a synergistic cytotoxic interaction exists between H202 and purified peptide defensins. These results are important in understanding the potential and the

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mechanism by which PMNs can exert anti-tumor effects and also injure normal tissue during the course of pulmonary, renal, or rheumatological diseases. ACKNOWLEDGMENTS This work was supported by Grants CA 37 184, HL 35640, AI 22839, and AI 2293 1 from the National Institutes of Health, N 00014-86-K-0525 from the Office of Naval Research, and research funds of the Veteran’s Administration. The authors thank Miriam Seelig, Julia Talos, and Sylvia Harwig for excellent technical assistance and Erzsebet Huflinan for typing the manuscript.

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