CELLULAR

IMMUNOLOGY

128,503-5 15 (1990)

Inhibition of Polymorphonuclear Leukocyte Oxidative Metabolism by ExogenousPhospholipaseC LEO I. GORDON,’

CYNTHIA SCHMEICHEL, SHEILA PRACHAND, AND SIGMUND A. WEITZMAN

Northwestern University, Department of Medicine, Section of Hematology/Oncology, 303 East Chicago Avenue, Chicago, Illinois 6061I Received January 19, 1990; acceptedMarch 5, 1990 We studied the effectsof exogenous, purified phospholipase C (PLC) on neutrophil oxidative metabolism, lysosomal enzyme release and aggregation. We found that PLC inhibited 0; and HZ02 generation and oxygen consumption, but did not alter glucose oxidation via the hexose monophosphate shunt. In contrast, we found a striking stimulation of aggregation and release of the lysosomal enzymes lysozyme and &+lucuronidase. In experiments designed to further characterize the mechanism of the PLC effect on membrane activation we studied the effect of PLC on intracellular calcium concentration [Ca*‘li and found that PLC did not interfere with the fhILP-mediated rise in [Ca’+]i, suggestingthat its inhibitory effect on the respiratory burst does not involve inhibition of early signal transduction events. In addition, we found that PLC alone results in mobilization of intracellular Ca*+ stores, consistent with its stimulatory effect on aggregation and lysosomal enzyme release. 0 1990 Academic press, IX.

INTRODUCTION Human polymorphonuclear leukocytes (PMN), when triggered by any one of a number of agonists, can metabolize oxygen to reactive oxidants (1). These cells can take up oxygen at a rapid rate, and utilize a membrane-bound NADPH oxidase system which generatessuperoxide anion radical (0;) a one-electron reduction product. The NADPH oxidase requires flavin adenine dinucleotide (FAD) (2,3), and the reaction may be characterized as follows: NADPH + H+ + 202 --* NADP+ + 2H+ + 20;. Concomitantly, there is oxidation of glucose via the hexose monophosphate shunt, a process which provides NADPH as a source of electrons for the production of 0;. We (4) and others (5) have previously shown that certain cellular enzymes, produced by inflammatory phagocytes at sites of inflammation may regulate oxidative metabolism in PMN. Recently, Styrt et al. (6) have shown that exogenous bacterial phospholipase C (PLC) alters oxidative metabolism in bovine PMN, and suggestthat this enzyme could play a regulatory role in phagocyte-bacteria interactions. In our current studies, we examined the effectsof two forms of purified bacterial phospholi’ To whom reprint requests should be addressed. 503 0008-8749190$3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

504

GORDON

ET AL.

pase C, which is a membrane and cytosolic enzyme (7), on PMN oxidative functions (production of O;, H202, and oxygen consumption), and on glucose oxidation via the hexose monophosphate shunt. We also measured the effects of exogenous PLC on other membrane-dependent events, including aggregation and lysosomal enzyme release.In experiments designed to further characterize the mechanism of PLC effect on membrane activation, we measured intracellular Ca” concentration ([Ca”]i) in resting and fMLP-stimulated PMN exposed to PLC. MATERIALS AND METHODS Reagents Phorbol myristate acetate (PMA) was purchased from Consolidated Midland Corp. (Forrester, NY) and stored at -4°C at a stock concentration of 10 mg/ml in dimethylsulfoxide (DMSO), and used in a final concentration of lo- 100 rig/ml for 02 and H202 determination and 50 rig/ml for O2 consumption experiments. N-Formyl-methionyl-leucyl-phenylalanine (fMLP) was purchased from Sigma Chemicals, (St. Louis, MO) stored at a stock concentration of 1O-6M and used in a final concentration of lo-’ M. Isolation of Polymorphonuclear Leukocytes PMN were isolated as previously described (4). Briefly, 30-50 cc of blood was drawn into a syringe containing 1 u/ml of heparin and was allowed to sediment with one-half volume of 6.5% dextran and 0.98% sodium chloride. The supernatant was collected, the remaining red cells lysed by hypotonic lysis and the pellet was resuspended and layered onto a Ficoll-Hypaque gradient and centrifuged at 450g for 30 min. The pellet contains 95% PMN and was suspended in HBSS +0.2% human serum albumin (HBSS-HSA) or HBSS alone. Superoxide Generation Superoxide anion generation was determined by the cytochrome C assay as described by Goldstein et al. (8). PMN were incubated with varying concentrations of PLC. Superoxide generation was measured as super-oxide dismutase inhibitable reduction of cytochrome C at 550 nm using a molar extinction coefficient for this change in absorption of 2 1,000. Agonists for these experiments included PMA ( 10 rig/ml) or lMLP (lo-’ M). In some experiments 0; generation was measured in a continuous ( 1-min time points) assayat 550 nm using a Varians spectrophotometer. HMP Shunt Oxidation of glucose via the hexose monophosphate shunt was assayedby generation of 14C02from [ l-‘4C]glucose as previously described (9). Briefly, D[‘“C] glucose (New England Nuclear Corp., Boston) labeled at the l-carbon position was added to PMN at 2.5 X 10d6PMN/ml. PMA or PMA and PLC at various concentrations were added and 14C02was trapped by hyamine-impregnated filter paper and then counted in a scintillation counter (Beckman). Data were expressedas counts per minute.

INHIBITION

OF NEUTROPHIL

OXIDATION

BY PHOSPHOLIPASE

C

505

Lysosomal Enzyme Release Lysosomal enzyme releasewas measured by previously described techniques (10). Briefly, 2.5 X 10m6PMN/ml were incubated in media with varying concentrations of PLC, then stimulated with PMA ( 10 rig/ml) or IMLP ( 10m7M). Supernatant was then assayedfor lysozyme using the rate of lysis of micrococcus luteus as determined by decreasein absorbance at 450 nm. P-glucuronidase was measured after 18 hr of incubation using P-nitrophenol glucuronide as a substrate. Total cellular lysosomal enzyme content for both enzymes was measured after sonication of cells, and is considered to be 100%. Supernatant of resting and stimulated PMN are expressedas a percent of total cellular enzyme. PMN Aggregation PMN aggregation was measured by previously described methods (11). Briefly, PMN preincubated with cytochalasin B (5 pg/ml) suspended with or without PLC, were added with stirring to a cuvette of a standard platelet aggregometer which was modified for PMN aggregometry. Change in light transmission was recorded as a deflection of light and was measured from the time of stimulation with known aggregating agents PMA or FMLP with and without PLC. Hydrogen Peroxide Determination Measurement of hydrogen peroxide was determined by peroxidase catalyzed oxidation of phenol red as previously described (12). Briefly, PMN were suspended in buffered phenol red solution (PRS) containing 140 PM NaCl, 10 mM KC1 buffer (pH 7.0), 5.5 rruV dextrose, 0.28 mM phenol red, and 5.5 units/ml of horseradish peroxidase (HRPO). Cells were stimulated with agonist, then after 30 min incubation centrifuged and 10 ~1of 1 N NaOH was added to the supernatant. Samples were read at 6 10 nm against a blank of 1 ml of PRS to which 10 ~1 NaOH was added. This was compared to an H202 standard curve performed concomitantly. Results were expressedas nanomoles of H202/2 X 1Oe6PMN/30 min. Oxygen Consumption Oxygen consumption was measured using previously described methods (13). Briefly, cells were incubated in a Clark-type electrode (Yellow Springs Instrument Co., Yellow Springs, OH). The glassof the electrode chamber was siliconized. Oxygen consumption in the presence or absenceof PLC was measured after stimulation with PMA. Cells were incubated at 1 X lop7 in a total volume of 1.8 ml. PMA was added at a final concentration of 50 rig/ml and all experiments were done in the presence of HBSS with Ca*+ (1 mM), Mg*+ (1 mM), and 0.2% human serum albumin. Data were expressedas nanometer of oxygen consumed per 10m7PMN/min. Phospholipase C Preparation Two preparations of PLC were utilized. Chromatographically purified PLC type 14 from Clostridium welchii was purchased (Sigma Chemicals, St. Louis, MO) and dissolved in HBSS at a stock concentration of 1 mg/ml. Concentrations ranging from 2 pg/ml to 100 pg/ml were used in all experiments. In addition, partially purified

506

GORDON

ET AL.

PLC C. welchii, (United Biochemical) was further purified by previously described methods ( 14, 15) utilizing an affinity column (agarose-linked egg yolk lipoprotein) followed by separation on a G-100 Sephadex column and heretofore referred to as PLC G- 100. This preparation was purified to homogeneity asdefined by a single band on Coumassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both of the PLC preparations had a preference for phosphatidylcholine as substrate. When Sigma 14 PLC activity was measured (see below) there was loo-fold more activity for phosphatidylcholine (PC) than for phosphatidylinositol (PI); specific activity for PC = 144 pm/mg protein/min, for PI = 1.3 pm/mg protein/min. There was minimal activity when phosphatidylserine or phosphatidylethanolamine were used as the substrates. Since the results to be described were identical with either the G 100 preparation or the Sigma 14 preparation, all experiments described below were done utilizing the Sigma 14 preparation. Phospholipase C Activity PLC activity was measured as described by Ma&in et al. ( 16). Briefly, radiolabeled substrate (PC or PI) was prepared by mixing labeled with unlabeled substrate, and dried under N2 at 37°C. The substrate was then resuspended, and sonicated for 15 min. Aliquots (200 pA4, 0.1 PC13H) were added to 250 PL of 0.06 M Tris acetate buffer, which contained 5 PM CaC& and 0.5 mg/ml deoxycholate. Five microliters of protein (to be assayed)was added and the mixture incubated at 37’C for 30 min. The reaction was then terminated by addition of 1.5 ml of butanol/chloroform/ 12 N HCl (100: 100:0.6 v/v/v) and then 0.45 ml of 1.O M HCl. This was vortexed and centrifuged at 8OOg.Aliquots of the aqueous phase (250 ml) containing radioactive inositol phosphates were removed and counted by liquid scintillation. Measurement of [ Ca2+]i PMN were loaded with Fura- using a modification of the method described by Tsien et al. ( 17). In these experiments PMN were incubated in P-GCM buffer, which consisted of the following: 0.025 M piperazine-N,N’-(Zethanesulfonic acid) (Pipes), 0.11 M NaCl, 0.005 M KCl, 0.6 rmJ4CaC12, 1 mM MgCl* , and 0.04 M NaOH, pH 7.4, containing 5.4 mM glucose. PMN ( lo8 cells/ml in P-GCM) were incubated in the dark at 37°C with 25 M Fura- (acetomethoxy ester) for 22 min. The PMN suspension was then diiuted IO-fold with P-GCM and incubated in the dark at room temperature for an additional 30 min. After centrifugation at 200g for 8 min at 4°C the Fura- loaded PMN were resuspended at 5 X lo6 cells/ml in ice-cold PAGCM (P-GCM with 0.003% human serum albumin) and kept on ice until use. The [Ca2+]i changes in the Fura- loaded PMN were measured fluorometrically in an AmincoBowman spectrofluorimeter equipped with a thermostatted cuvette chamber. PMN (2 X lo6 cells/ml) were preincubated in a siliconized cuvette at 37°C for 5 min with 5 pg/ml cytochalasin B in PAGCM buffer; the cells were then incubated for an additional 4 min at 37°C with buffer or the PLC. The fMLP (50 ti) was added to the cuvette and the change in maximum and minimum fluorescence values over time were determined by the addition of 80 ~1of 1% (v/v) Triton X-100 and 40 ~1 of 0.5 M Tris, 0.5 M EDTA (pH lo), respectively, to 2 ml of cell suspension. Autofluores-

INHIBITION

OF NEUTROPHIL

OXIDATION

BY PHOSPHOLIPASE

C

507

cence of unloaded PMN was negligible. The [Ca2+]i was calculated from the fluorescence values as described elsewhere ( 17). RESULTS Inhibition ofReactive Oxygen Metabolite Production by PLC We found that preincubation of PMN with PLC inhibited 0; and H202 production after stimulation with PMA. Figure 1a shows inhibition of 0; generation by PLC at varying concentrations after PMA exposure. Similarly, there was inhibition of H202 generation by PLC preincubation (Fig. 1b) after PMA stimulation. The effects were not the result of decreasedcellular viability, as even at concentrations as high as 100 Kg/ml viability was >90% as measured by trypan blue dye exclusion and LDH release. PLC alone did not influence PMN 0; generation, and addition of PLC concomitant to or after stimulation with PMA caused no inhibition of 0; generation. In order to establish the kinetics of the inhibitory effects of PLC on PMN respiration we measured PMA-stimulated 0: generation using a continuous (1-min time points) assayat an absorbance of 550 nm. We found that PLC caused a dose-dependent inhibition of 0, generation following both PMA (Fig. 2) and fMLP (Fig. 3) stimulation. Efects of PLC Preincubation on PMN Oxygen Consumption We found that PLC could, when preincubated with PMN, inhibit oxygen consumption after stimulation with PMA (Table 1). We observed a 40-50% inhibition of oxygen consumption with PLC preincubation of 15 min, but no inhibition was seen if PLC was added following PMA activation (data not shown). Efects of PLC on Hexose Monophosphate Shunt Activity We measured C- 1-glucose oxidation with PMA stimulation after PLC preincubation. As shown in Table 2, we found that PLC preincubation caused no inhibition of 1-glucose oxidation (measured as 14C02) following stimulation with PMA. PMN were preincubated with PLC for 15 min prior to PMA activation. We also found that PLC and PMA added simultaneously did not inhibit glucose oxidation. Efects of PLC on PMN Aggregation and Lysosomal Enzyme Release Exogenous PLC, when added to human PMN, caused striking aggregation (Fig. 4), exhibiting aggregation curves similar to those seenwith known aggregantsfMLP plus cytochalasin B. Further, addition of PLC to PMN resulted in releaseof the lysosomal enzymes lysozyme and &$curonidase (Table 3). Thus aggregation and enzyme release occurred with addition of PLC alone and did not require another activator. Addition of agonist to PMN following PLC incubation resulted in increased degranulation (60~80%) when compared to PLC alone or tMLP alone (data not shown). Eflects of PLC on PMN Intracellular Ca’+ To determine the point in the signal transduction pathway at which PLC was acting, we examined the integrity of the fMLP-stimulated calcium response following preincubation. In PMN preincubated with 70 pg/ml of PLC, a concentration which

a

31 n=4

16 n-4

I

pg/ml

; 4 ,-

63 n-5 PLC

b

T

+ 01 24

8

31

16 Fg/ml 5OR

PLC

a3

INHIBITION

OF NEUTROPHIL

OXIDATION

BY PHOSPHOLIPASE C

509

35 6 fj 3 $ L

30 25

0 0 5

20

5E

10

'N

0

15

5 0 1

2

3

4

6

6

10

12

14

Time (mid FIG. 2. PMN are incubated with varying concentrations of PLC, then stimulated with PMA (10 rig/ml). Superoxide generation, measured as SOD inhibitable reduction of cytochrome C, is assayedat I-min time intervals in a Varians spectrophotometer. There is a dose-dependent inhibition when PLC-incubated PMN are compared with controls. Control (a), PLC 12.5&ml (0), PLC 25 &ml (0) PLC 100 &ml (0).

resulted in maximal inhibition of respiratory burst activity (see above), IMLP (50 nM) increased [Ca2+]i as measured by Fura 2 to 280 nM, a response fourfold greater than PMN exposed to NLP without PLC preincubation (Fig. 5). The kinetics of the fMLP stimulated increase in [Ca2+]i were identical to that observed in PMN stimulated without PLC. PLC alone also increased [Ca’+]i, so that within 5 set of PLC addition there was an increase in [Ca2+]ifrom a basal level of 86 to 182 nM. This was not inhibitable by EGTA (Fig. 5). DISCUSSION We have demonstrated that exogenous bacterial phospholipase C (PLC) inhibits superoxide and hydrogen peroxide generation induced by PMA and IMLP. The effects of PLC on 0; and H202 generation were not due to scavenging of generated 0; and H202. This was confirmed by experiments where PLC was added immediately following or concomitant to addition of agonist. In these instances, we observed no inhibition. We also found that PLC inhibited oxygen consumption in PMN by approximately 45%, but that addition of PLC following stimulation by PMA was not inhibitory. In an attempt to uncover the mechanism of the observed inhibition of 0; and H202 by PLC, we examined the kinetics of the inhibitory effect. Prior reports describe a lag phase of measureable reduction of cytochrome C following PMA stimulation ( 18). We found that PLC caused a dose-dependent inhibition of PMN activation and the dose-response curve for PLC inhibition was similar for the two agonists. Since PMA and fMLP activate the respiratory burst by different mechanisms at different site(s) on the PMN membrane, the similarity of the dose-response curves suggests

FIG. 1. (a, b) PMN are incubated for 15 min with varying concentrations of PLC, then stimulated with PMA (10 rig/ml). Superoxide generation (a) measured as SOD inhibitable reduction of cytochrome C is assayed as described under Materials and Methods. Hydrogen peroxide generation (b) is measured by peroxide catalyzed oxidation of phenol red against a standard curve as described under Materials and Methods.

510

GORDON

ET AL.

5

Time (mid FIG. 3. PMN treated as in Fig. 2, but cells are preincubated with cytochalasin B 5 &ml with fMLP (IO-’ M). There is again dose-dependent inhibition of superoxide generation.

then stimulated

that the site of PLC action is at or near the common pathway for respiratory burst activity, the NADPH oxidase. We also found that PLC caused release of the lysosomal enzymes lysosyme and ,& glucuronidase, and stimulated aggregation of PMN to a level that was comparable to that seen with known PMN agonists. These stimulatory effects on PMN by PLC were contrary to what might have been expected from the respiratory burst data, and suggestperhaps that there are different mechanisms of action of exogenous bacterial PLC on different PMN functions. To determine the point in the signal transduction pathway at which exogenous PLC might be acting, we examined the integrity of the fMLP-stimulated calcium response, an early signal transduction event. In PMN preincubated with PLC at con-

TABLE

1

Effects of PLC on O2 Consumption”

Addition None PLCb

O2 consumption (niii/lO’ PMN/Min) 18.9k 1.1 10.9 f 0.97’

’ Measured as f SEM of two experiments each performed in duplicate, using PMA 100 rug/ml as stimulus. b PLC 63 &ml is preincubated with PMN for 10 min. c P < 0.05 by two-tailed Students t test.

INHIBITION

OF NEUTROPHIL

OXIDATION

BY PHOSPHOLIPASE C

511

TABLE 2 PLC Does Not Inhibit PMA Stimulated Oxidation ofD-[%]Glucose” Addition to PMN

cpm 19.5 + 223.6 + 252.5 f 235.9 f 45.1 +

HBSS PMA PLC’ + PMA (PLC + PMA)d PLC’

1.67” 19.50 25.02 12.97 6.50b

a Measured as k SEM of two experiments each performed in quadruplicate (i.e., eight determinations) using PLC 63 &ml and PMA 10 rig/ml. bThe difference is not statistically significant by Students t test. ’ PLC (63 PaJrnl) preincubated with PMN for 15 min followed by PMA. d PLC and PMA added simultaneously. ePLC alone without PMA stimulation.

centrations which inhibited respiratory burst activity, fMLP resulted in an increase in [Ca2+]i(Fig. 5). The kinetics of the fMLP-stimulated increase in [Ca2+]iwas identical to that observed in PMN stimulated without PLC. When PLC alone was added to PMN, there was an almost immediate rise in [Ca2+]i. Within 5 set of PLC addition there was an increase in [Ca2+]i from basal levels. There was no inhibition of this effect by EGTA, suggestingthat this rise was due to mobilization of Ca2+from intracellular stores rather than from influx of extracellular Ca*+ due to disruption of the PMN membrane. These data make it unlikely that PLC is exerting its inhibitory effect on the respiratory burst by inhibiting early signal transduction events. Furthermore, the observation that PLC induces a rapid rise in [Ca2+]i without fMLP is consistent with its observed stimulator-y effects on PMN degranulation and aggregation, and suggest that exogenous bacterial PLC may be operating by more than one mechanism. All of these effectswere observed for different preparations of PLC, suggesting that contaminants were likely not responsible for the various effects. The PLC used

PLC 50 pghl

/

AT

FIG. 4. Phosphohpase C, when added to human PMN, resulted in a dose-dependent aggregation over a IO-min time period.

512

GORDON

ET AL.

TABLE 3 Effects of PLC on PMN Lysosomal Enzyme Release” 5%Release Addition to PMN

Lysozyme

@-Glucuronidase

Cyto B + F’MLPb PLC’63 pg/mld Noned

38.6 k3 (n=9) 36.0 k4.3(n = 6) 6.71 f 1.3 (n = 6)

27.3 +3.3(n=S) 37.02 +4.6(n = 5) 11.17+I.l(n=6)

a Data are expressed as percentage of total cell sonicate. b PMN are incubated with cyto B for 5 min and F’MLP lo-’ Mfor 15 min. ’ PLC are preincubated with PMN for 20-30 min. d P = 0.05 by Students t test.

in these experiments was derived from C. welchii and preferred phosphatidylcholine as substrate. Furthermore, there was no loss of cellular viability by trypan blue exclusion or by LDH release at doses up to 100 pg/ml for at least 30-40 min of incubation. In an attempt to elucidate the mechanism of the observed inhibition of 0; and H202 by PLC, we sought to examine the effects of PLC on glucose oxidation via the hexose monophosphate shunt, the system which, in PMN, is responsible for genera-

r

FMLP

5’ I+,

, 0

5

I

Minutes I 30

Addiii

3’ 1

t 60

h

Minutes

I 0

I 5

I

I

30

I

I

00

FIG. 5. PMN preincubated with cytochalasin B (5 &ml) for 5 min are then treated with PLC (70 pg/ ml). Intracellular calcium concentration ([Ca*‘]i) is then measured as described under Materials and Methods. There is a striking rise in [Caz+li within 5 set of PLC addition (O), and this was not inhibitable by EGTA (w). At 4 mitt, PMN are then exposed to fMLP and [Ca2+li measured. There is a fourfold greater increase in [Ca*+k in PLC-treated cells (0) than in controls (0) without PLC but the kinetics of the rise in [Ca2+li were identical.

INHIBITION

OF

NEUTROPHIL

OXIDATION

BY PHOSPHOLIPASE

C

513

tion of NADPH electrons. We found that preincubation of PMN with PLC under conditions identical to those described earlier resulted in no inhibition of glucose oxidation (measured as i4C02) compared to controls with no PLC. Further, PLC alone caused no oxidation of glucose. These results are surprising, since HMP shunt activity during the respiratory burst is felt to be secondary to accumulation of intracellular H202 and depletion of NADPH, both occurring as a consequence of NADPH oxidase activity. We cannot easily reconcile these data in light of our other observations, unless the timepoint of measurement of 14C02 (15 min) was too late to detect an inhibitory effect. We observed differences in the degree of inhibition of H202 and 0; and differences in the degree of inhibition of total oxygen consumption and reduced oxygen species. We cannot readily account for the quantitative differences observed from the data, but may speculate that this difference could be due to selective intracellular destruction of one species over the other. It is unclear by what mechanism PLC is causing the effects observed here, although our data point to a site in the membrane signal transduction pathway between the receptor complex (for fMLP) and the NADPH oxidase. PLC, attached to cell membranes or in cytosol, is known to hydrolyze inositol phosphates to second messengers inositol triphosphate and diacylglycerol(l9). PLC activation is mediated by G proteins (20) and these events accompany signal transduction, including events associated with the respiratory burst (21). Indeed, there are data extant which implicate products of PLC activation in “priming” of the respiratory burst in PMN (22). Because the PLC used in these studies preferred phosphatidylcholine (PC) (and not phosphatidylinositol (PI)) as substrate we cannot determine whether specific inositol pathways were involved. In fact, the mechanism of the effects observed in our experiments may not be related to the second messenger system at all, but may reflect alteration of the configuration or orientation (steric effects) of components of the electron transport system, perhaps induced by hydrolysis of certain membrane phospholipids. Thus we cannot be certain from our data whether the mechanism of PLC action involves second messengers. If not, then the steric effects that may be induced by PC preferring PLC could be important. If so, recent experiments showing that DAG accumulates by the action of PC preferring PLC as well as PI preferring PLC make an understanding of the effects of exogenous PLC relevant. Besterman et al. (23) have shown that DAG and phosphocholine can be generated in a variety of cell types following stimulation with phorbol diesters, serum and PDGF. These data indicate that at least some of the DAG is derived from activation of a PC preferring PLC. Similarly, Irving (24) has shown that a PC preferring PLC can hydrolyze PC in rat liver cells and contribute to hepatic DAG levels and influence protein kinase C activity. More recently, Grillone (25) has provided evidence for release of arachidonic acid as well as DAG and phosphocholine from smooth muscle cells via a vassopressin receptor, activation of which in turn activates both PC and PI preferring PLC. Further elucidation of the potential role of PC as substrate for the production of second messengers in PMN appears warranted. For some agonists, NADPH oxidase activation is related to protein kinase C translocation from the cytosol to the membrane (26), a process that can be accomplished by diacylglycerol, a product of PLC-mediated hydrolysis of phosphoinositides (27). For this reason, our observations that PLC inhibits 0; generation is surprising, since

514

GORDON

ET AL.

generated diacylglycerol would be expected to activate protein kinase C and initiate cellular responses (including 0; generation). It is possible, however that a phosphatidylcholine preferring PLC acts in some other way on PMN membranes to inhibit PKC activation, and thus serve as a termination or negative feedback signal. The recent experiments by Jones et al. (28) support this concept, since they show that exogenous PLC, incubated for long periods with mouse epidermal (HEL-37) cells, inhibit PKC activity by inhibiting cytosol-membrane translocation. A similar system may be operative in PMN. Nevertheless, the influence of an exogenous enzyme on intracellular events is not clear, so the results as they relate to changes in intracellular messengers or changes in intracellular compartments must be interpreted with caution. There have been previous studies examining the effects of exogenous PLC on PMN function. Rossi and his group demonstrated stimulation of respiratory burst activity by a PLC preparation in guinea pig neutrophils and postulated involvement of products of the phosphoinositide pathway as mediators of this effect (29, 30). However, Styrt et al. (6) recently found no effect of Clostridium perfringens or Bacillus cereus PLC on bovine PMN 0, production, but did find a small priming effect when the PMN were subsequently stimulated with NaF. Interestingly they found inhibition of oxygen consumption after particulate stimulation of PMN, a finding similar to the observations described herein. We found no direct effect of two preparations (purified to homogeneity by two methods and preferring phosphatidylcholine as substrate) of C. welchii PLC on human 0; or H202 generation, but found striking dose-dependent inhibition that was most pronounced during the first 8-10 min following stimulation by PMA or IMLP. Some of these differences may be due to the purity of the preparations utilized, or may reflect differences in human versus bovine or guinea pig PMN. In summary then, we describe inhibition of 0; and H202 generation in human PMN by exogenous PLC following stimulation with soluble stimuli, yet find no alteration in other membrane-dependent functions. We also describe a striking and immediate increase in intracellular Ca*+ in PLC-exposed PMN, but no inhibition of NLP stimulated Ca*+ mobilization. These data suggest that exogenous bacterial PLC (preferring phosphatidylcholine) effects PMN respiratory burst activity by a mechanism that is likely to involve a site in the membrane between the fMLP receptor complex and the NADPH oxidase. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Weiss, S. J., Acta Phys. Stand. 126,9, 1986. Babior, B., J. Clin. Invest. 73,599, 1984. Babior, B., Blood64,959, 1984. Gordon, L. I., Douglas, S. D., Kay, N. E., Yamada, O., Osserman, E. F., and Jacob, H. S., J. C/in. Invest. 64,266, 1979. Gray, J. C., Beckman, S. K., Brash, A. R., Oates, J. H., and Lukens, J. N., Blood 64,780, 1984. Styrt, B., Walker, R. D., and White, J. C., J. Lab. C/in. Med. 114,51, 1989. Majerus, P. W., Connoly, T. M., Deckmyn, H., Ross, T., Brass, T. E., Ishii, H., Bansal, V. S., and Wilson, D. B., Science 234, 15 19, 1986. Goldstein, I. M., Roos, D., Kaplan, H. B., and Weissman, G., J. Clin. Invest. 56, 1155, 1975. Keusch,G. T., Infec. Immun. 5,414, 1975. Goldstein, I. M., Hoffstein, S. R., and Weissman, G., J. Cell Biol. 66,647, 1975. Craddock, P. R., Hammerschmidt, D., White, J. G., Dalmasso, A. P., and Jacob, H. S., J. C/in. Invest. 60,260, 1977. Pick, E., and Keisari, Y., J. Immunol. Methods 38, 16 1, 1980.

INHIBITION

OF NEUTROPHIL

OXIDATION

BY PHOSPHOLIPASE

C

515

13. McPhail, L. C., Henson, P. M., and Johnston, R. B., J. Clin. Invest. 67,710, 1981. 14. Takahashi, T., Sugahara, T., and Ohsaka, A., In “Methods in Enzymology” (J. M. Lowenstein, Ed.), Vol. 7 1, pp. 720-725, 198 1. 15. Cautrecasas, P., Proc. Natl. Acad. Sci. USA 61,636, 1968. 16. Mackin, W. M., and Stevens, T. M., J. Leuk. Biol. 44,8, 1988. 17. Tsien, R. Y., Pozzan, T., and Rink, T. J., J. Cell. Biol. 94,325, 1985. 18. Newberger, P. E., Chovaniec, M. E., and Cohen, H. J., Blood%, 85, 1980. 19. Smith, C. D., Lane, B. C., Kusaka, I., Verghese, M. W., and Snyderman, R., J. Biol. Chem. 260,5875, 1985. 20. Smith, C. D., Cox, C. C., and Snyderman, R., Science 232,97, 1986. 2 I. Smith, C. D., Uhning, R. J., and Snyderman, R., J. Biol. Chem. 262,6 121, 1987. 22. Bass, D. A., Gerard, C., Olbrantz, P., Wilson, J., McCall, C. E., and McPhail, L. C., J. Biol. Chem. 262,6643,1987. 23. Besterman, J. M., Duronio, V., and Cautrecasas, P., Proc. Natl. Acad. Sci. USA 83,6785, 1986. 24. Irving, H. R., and Extort, J. H., J. Biol. Chem. 262,3440, 1987. 25. Grillone, L. R., Clark, M. A., Godfrey, R. W., Stassen, F., and Crooke, S. T., J. Biol. Chem. 263,2658, 1988. 26. Cox, J. A., Jeng, A. Y., Sharkey, M. A., Blumberg, P. M., and Tauber, A. I., J. C/in. Invest. 76, 1932, 1985. 27. Cox, R. I., Daugherty, R. W., Garorg, B. R., J. Immunol. 136,4611, 1986. 28. Jones, M., and Murray, A. W., Biochem. Biophys. Res. Commun. 136, 1083, 1986. 29. Patriarca, P., Zatti, M., Cramer, R., and Rossi, F., Ll$ Sci. 9, 84 1, 1970. 30. Grzeskowiak, M., Della Blanca, V., DeTogni, P., Papini, E., and Rossi, F., Biochem. Biophys. Acta 844,81, 1985.

Inhibition of polymorphonuclear leukocyte oxidative metabolism by exogenous phospholipase C.

We studied the effects of exogenous, purified phospholipase C (PLC) on neutrophil oxidative metabolism, lysosomal enzyme release and aggregation. We f...
825KB Sizes 0 Downloads 0 Views