Neutrophil signal transduction in Met-enkephalin modulated superoxide anion release

Neuropeptides (1996) 30 (2), 193-201 © PearsonProfessionalLtd 1996

Neutrophil signal transduction in Met.enkephalin modulated superoxide anion release H. Haberstock ~, T. MarottP, H. Banfic 2 1Rudjer Boskovic Institute, Department of Experimental Biology and Medicine, Laboratory for Biological Response Modifiers, Zagreb, Croatia. 2University of Medicine, Department of Physiology, Zagreb, Croatia

Summary The present study explored the involvement of signal transduction system(s) in Met-enkephalin (MENK) modulated superoxide anion (O~) release from human neutrophils. This opioid pentapeptide stimulated the O~ release in all samples if present at 10.8 M concentration while in lower concentrations the stimulatory concentration was donordependent. The most abundant product of MENK degradation, Tyr-Gly-Gly (TGG), suppressed O~ release over a wide range of concentrations (10-12-10-8 M). MENK induced O~ release was associated with a dose-dependent increase of diacylglycerol (DAG) concentration and protein-kinase C (PKC) transiocation to the neutrophil membranes, with an increase of cytosolic Ca++, and could be abolished by H7, a PKC inhibitor. On the contrary, the suppressive effect of TGG was not associated with alteration of DAG concentration in neutrophil membranes. Superoxide anion release induced by low concentrations of MENK (1012-10-~° M), could be blocked by NDGA, an inhibitor of the lipooxygenase pathway. We concluded that MENK-induced OZ release results mainly due to DAG/PKC pathway activation, although other secondary messengers might be involved.

INTRODUCTION

Phagocytes produce large amounts of reactive oxygen species, such as superoxide anion (Oj), as part of their microbicidal armament in the process of respiratory burst catalyzed by the NADPH-oxidase. Microbicidal and cytotoxic activity of the phagocytic cells can be modulated by hormones of neural origin, including the opioid peptides. ~,2In our hands, Met-enkephalin (MENK) modulated the 0 2 production by human polymorphonuclear neutrophils. 3However, the mechanisms by which opioids regulate the cytotoxic activities of the phagocytes have not been sufficiently defined. The existence of 6, g and K opioid receptors on human PMNs and monocytes has been confirmed. 4-~ These opioid receptors, coupled to the G-protein, use adenylate

Received 23 October 1995 Accepted 10 February 1996 Correspondence to: H. Haberstock, Institute Rudjer Boskovic, Department of Experimental Biology and Medicine (EBM), Bijenicka 54, 10000 Zagreb, Croatia.

cyclase as a second messenger? Childers 7 demonstrated that g and 6 agonists open the potassium channels, and K agonists close the calcium channels through direct interaction between the G-protein and the ion channels. Some p and K receptor agonists, as well as the naturally occurring opioid pentapeptide MENK, increase the level of free cytosolic calcium in human B cell lines in a dose-dependent manner, s The activation of the neutrophil NADPH-oxidase, capable of catalyzing the single electron reduction of molecular oxygen to OL may involve several transduction pathways. The most examined pathway is the phospholipase C-mediated phosphatidylinositol 4,5-biphosphate hydrolysis, leading to the generation of inositol 1,4,5triphosphate. This, in turn, mediates the increase in intracellular calcium and diacylglycerol, a PKC activator. Although sufficient data exist about signal transduction pathways coupled to the induction of the oxidative burst by various agonists, signal transducers involved in MENK modulated superoxide anion release in human PMNs are still incompletely known. The aim of the present study was to examine the signal transduction pathways involved in the MENK action on Oi production in human neutrophils. 193

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MATERIALS AND METHODS Chemicals

Dextran T-500 was from Pharmacia, Sweden. Superoxide dismutase (SOD), cytochrome C, MENK, Tyr-Gly-Gly (TGG), phorbol 12-myristate 13-acetate (PMA), N-formylL-methionyl-L-leucyl-L-phenylalanine (fMLP), (1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine) (H7), nordihydroguaiaretic acid (NDGA), Quin 2-AM, ethylene glycol-bis ({3-aminoethyl ether (EGTA) and Triton X-100 were from Sigma, USA. Isolation of human neutrophils

Blood from adult healthy donors was collected into plastic universal tubes containing 10 u/ml of preservativefree heparin. The blood was mixed with 3% dextran (ml dextran/ml of blood) and left 45-60 rain at room temperature. The leucocyte-rich plasma was collected and contaminating red blood cells were removed by hypotonic lysis for 20 s in cold deionized water. The PMN cells were isolated on Ficoll-Hypaque by using Boyum's technique. 9 PMNs were suspended in phenol-free Hank's balanced salt solution (HBSS). This procedure typically yielded preparations containing more than 95% PMNs. Assay for superoxide anion (O;) release

Superoxide anion was measured as SOD inhibitive reduction of cytochrome C using the modification of the method of Johnson et al2 ° Optical density of the supernatant was determined spectrophotometrically at 550 nm. The concentration of reduced cytochrome C was calculated using the formula E550nm = 2.1 x 104 M -1 cm -1. Quantification of free cytosolic Ca 2*

Human PMN leucocytes were loaded with Qiun-2AM by using a modification of the method described by Tsien et al." Replicate suspension of 2x10 z PMNs/ml HBSS were incubated at 37°C with 2xlO -~ M Qiun-2AM for 15 min in the dark, and then for a further 20 min at room temperature. After washing several times with HBSS, the cell pellets were resuspended at approximately l xlO 7 PMN/ml and stored on ice until used for the fluorescence measurements. The fluorescence of Quin-2AM containing PMNs was recorded with a Perkin-Elmer LS 50 spectrofluorimeter equipped with a magnetic stirrer under the cuvette holder. The monochromators were set at 339 nm for the excitation with a 3 nm slit, and at 492 nm for the emission with a 20 nm slit in the ratio mode. PMNs containing Quin-2AM were pelleted for 15 s in a Beckman microfuge, resuspended in 2 ml of fresh HBSS at 37°C and stored in the dark for 15 min. This 15-min Neuropeptides (1996) 30(2), 193-201

incubation permitted Ca 2+ to reach a steady-state level. The fluorescence then was quantified for 3 rain before and 10 min after the introduction of a stimulus. The maximum and minimum fluorescence signals were determined for each preparation of cells by the addition of 20 gl of 1% (v/v) Triton X-100 in distilled water to 2 ml cell suspension followed by 40 gl of 0.5 M Tris, 0.5 M EGTA (pH 10). The autofluorescence was determined under identical conditions using an equal number of cells without Quin-2AM in 2 ml HBSS. The autofluorescence value, which accounted for 15% or less of the maximum fluorescence value, was subtracted from the fluorescence value of Quin-2AM-containing neutrophils, and the corrected values were used to calculate the intracellular calcium ion concentrations.n

Isolation of neutrophil membranes

PMNs (5x106) were homogenized in a Braun homogenizer at 1200 r.p.m., passing the pestle up and down 20 times. The homogenate was centrifuged in Sorval ultracentrifuge for 1 h at 105 000 g a n d 4°C. The supernatant (cytosolic fraction) was removed, and the pellet (membrane fraction) was resuspended in 200 gl of cold HBSS and stored at -70°C until the measurement of DAG and PKC levels. Protein content of each tube was determined by the method of Bradford. 12

Measurement of DAG accumulation in neutrophil membranes

DAG was extracted using 0.75 ml chloroform/methanol (1:2). Further extraction was performed as described by Folch et al. 13 After the lipids were dried, DAG was dissolved in 0.5 ml of chloroform and loaded on a silica acid column (0.5 ml made in a Pasteur pipette), eluted with 1 ml of chloroform, and dried once again. The mass measurement for DAG was performed as follows: the dried lipid was dissolved by the addition of 20 gl of CHAPS (9.2mg/ml) and sonicated at room temperature for 15 s. After the addition of 80 gl of buffer (50 mM Tris-acetate, 80 mM KC1, 10 nM magnesium acetate, 2 nM EGTA, pH Z4), the reaction was started by the addition of 20 gl of DAG Mnase enzyme followed by 80 gl of buffer containing 5 gM ATP and 1 gCi (32p)ATP. After 1 h at room temperature the reaction was stopped by the addition of 750 gl of chloroform/methanol/HC1 (80:160:1). PtdOH was extracted as described by Folch et a113 and chromatographed on oxalate (l%)-sprayed TCL plates using the following solvent system: chloroform/methanol/concentrated ammonia/water (45:35:2:8, by vol). 14 After autoradiography, the spots corresponding to PtdOH were scraped off and their 32p content was determined by © Pearson Professional Ltd 1996

Neutrophil signal transduction in MENK modulated 0 d release

scintillation counting. Using the same procedure with known concentrations of DAG the standard curve was observed. Western blotting with anti-PKC antibody

After centrifugation in ultracentrifuge (Beckman) the pellet (the membrane fraction of 5x106 PMN) was resuspended in 300 gl of final buffer (10 mM Tris-HCl (pH 8.0), 5 mM MgC12, 0.32 M sucrose, 5 mM [3-mercaptoethanol and 1 nM EGTA). 50 gg of membrane fractions were subjected to electrophoresis on 10% SDS gels. Transfer to nitrocellulose was achieved using 10 nM sodium carbonate, 3 mM sodium bicarbonate buffer pH 9.9 for 1.5h. The blot was blocked in 20 mM Tris pH Z4, 0.5 M NaC1, 5% BSA, 0.2% Tween 20, followed by incubation with primary antibody (1:1000) overnight 4°C in the above buffer. After incubation with an anti-rabbit lgG conjugated to horseradish peroxidase (Sigma, 1:2000 at 4°C), visualization was performed using EeL systems (Amersham, USA). Statistics

The data were analyzed by Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney U-test for 2 samples. The level of significance was set at 5%.

195

Protein kinase-inhibitor (H7) counteracts MENKinduced O~ release from the neutrophils

Neutrophils from 6 donors were incubated with proteinkinase C (PKC) inhibitor H7 (2 x l0 -4 M) for 10 min at 37°C before the addition of MENK (10-12-10 -8 M) (Fig. 3). H7 diminished the stimulatory effect of 10-s M MENK (P
In order to examine whether the stimulatory effect of MENK or the suppressive effect of TGG are mediated by diacylglycerol (DAG) as the second messenger, neutrophils from 4 (out of 6) donors were exposed to 10-1210-8 M MENK or TGG for 30 min at 37°C. Control cells were incubated in medium. DAG concentration was measured in isolated membrane fractions. As shown in Figure 4, MENK significantly elevated the level of DAG in a dose-dependent manner. On the contrary TGG did not effect the level of DAG whichever concentration examined.

RESULTS MENK stimulates and TGG suppresses O~ release by human neutrophils

MENK causes PKC translocation to neutrophil membranes

Neutrophils from 6 healthy donors were incubated with 10-12-10.8 M MENK or TGG and cytochrome C (1 mg/ml) for 30 min at 37°C. Control cell samples of each donor were incubated with medium. The control, MENK- and TGG-treated samples, differed statistically: at 10-12 M, F(2,18) = 12.2, P = 0.02; at 10-12 M, F(2,18) = 11.8, P = 0.002; at 10-l° M F(2,18) = 12.7, P = 0.001; at 10-9 M, F(2,18) = 12.9, P = 0.001; and at 10.8 M, F(2,18) = 12.9, P = 0.001. In all samples and in all concentrations, TGG suppressed Of release as compared to O~ production in control, untreated neutrophils (at 10-12, 10-11, 10-s° and 10-9 M, P = 0.002; and at 10.8 M P = 0.007) (Fig. 1). On the contrary, MENK significantly stimulated O~ release in all samples if present in high concentration (10.8 M, P = 0.007); in the lower concentration range (10-12-10 -9 M) the stimulatory effect was variable, donor-dependent, and not statistically significant for the group. The stimulating effect of 10-8 M MENK was less pronounced but comparable to that of IO-~M fMLP, while 50 ng of PMA proved to be a much stronger inducer of O~ release (Fig. 2).

In order to demonstrate whether MENK-induced stimulation of Of release is associated with PKC as the second messenger, the level of PKC was determined in the membranes of neutrophils treated with 10-8 M MENK (Fig. 5). In neutrophfl samples of 3 donors, MENK induced PKC translocation to the cell membranes.

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MENK induced changes in free cytosolic calcium

Quin-2AM was used to determine the effect of MENK on the level of free cytosolic calcium in h u m a n neutrophils. The exposure to various MENK concentrations (1012-10-8 M) induced a large increase in free cytosolic calcium (Fig. 6). However, after 30 s a sharp decline occurred, and after 50 s the calcium level returned to the baseline level of control, untreated cells. The calcium increase caused by fMLP, as a positive control, was more rapid, reaching the maximum within 20 s and remaining at the high level for 50 s. Thus, MENK and fMLP seem to have different patterns in causing the increment in free cytosolic calcium. Neuropeptides (1996) 30(2), 193-201

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Fig. 1 The effect of M E N K or T G G (10-12-10 ~ M) on 0 2 production by human neutrophils. The data of O~- release by PMNs of 6 donors treated with various M E N K concentrations are expressed as percentage of O~ release from their control, unstimulated PMNs (100%) Each point represents a mean of a given dose of either M E N K (---) or T G G (...) in a particular donor tested in triplicate. */:'<0.01 vs own baseline value control vs T G G 10 12, 10 -11, 10 "1° and 10 .9 M P =0.002 control vs T G G 10 `8 P=0.007 control vs 10 -12, 1011 10 -1° and 10 .8 M M E N K NS control vs M E N K 10 -8 M P = 0:007

Neuropeptides (1996) 30(2), 193-201

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Neutrophfl signal transduction in MENK modulated O~ re~ease 197

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© Pearson Professional Ltd 1996

Neuropeptides (1996) 30(2), 193-201

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Lipooxygenase inhibitor of NDGA abrogates MENKinduced O~ release in human neutrophils In order to examine whether the effect of lower MENK concentrations might be mediated through the lipooxygenase pathway, neutrophils were pretreated with lipooxygenase inhibitor NDGA (10.5 M) for 10 min at 37°C before MENK (10-12-10 10 M) (Fig.7). The stimulatory effect of MENK was donor-dependent, i.e. in 2 samples (B,C) it was seen with 10-l° M MENK, while in 1 donor (A) in 10 ~2and 10-21 M. However, each stimulatory effect of MENK concentration was abrogated with NDGA. Neuropeptides (1996) 30(2), 193-201

As already demonstrated, 15 MENK stimulated 0 2 release in human PMN in a donor-dependent manner regarding the effective concentrations. However, in all donors tested in this study, O; release was consistently stimulated by 10-8 M MENK. Our previous results indicate that MENK-modulated O~ release is mediated through pertussis toxin-sensitive G-protein. 16 These observations have been confirmed with literature data showing that MENK in lower concentrations acts through the opioid receptors and guanilate cyclase.2 The aim of the present work was to further examine the signal transduction mechanisms involved in MENK effect upon the free radical release. We have tried to define whether MENK action on 0 2 release includes some classical signal transduction sequences shared by the chemoattractants; diacylglycerol, protein-kinase C and free cytosolic calcium. Our study has demonstrated that 10-12-10-8 M MENK increased the DAG level in the neutrophil membranes in a dose-dependent manner. Opposite to that, the main degradating product of MENK hydrolysis by enkephalinase, the tripeptide Tyr-Gly-Gly suppressed O; release, but the effect was not associated with changes in DAG level in the membranes. Thus, DAG seems to play an important role in MENK-induced stimulation of O; release, whereas the suppressive effect of TGG probably occurs through other signal transduction patterns. Together with our previous study about enzymatic degradation of MENK and the relevance of intact enkephalin molecules in stimulating the O; release, lz these observations may add to the possible explanation © Pearson Professional Ltd 1996

Neutrophil signal transduction in MENK modulated O~ release

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Fig. 6 MENK elevates the level of free cytosolic calcium in the neutrophils. The level of free cytosolic calcium in control, untreated PMNs is presented as 100% (range 110-130 nmol). The changes in free cytosolic calcium induced by MENK ( _ _ ) or fMLP (...) used as a positive control are expressed as percentages of the control level. The data from 1 of 3 experiments with similar results are shown.

of the donor-dependent modulation by opioid peptides observed by us ~6and others. TM Positive or negative adjustments of the immune functions could accrue from opposite signals generated by opioid peptides and their active degradation fragments and from non-uniform signal transmission. This view is in accordance with the results of Ohtsuka et al,19 who showed the relevance of diacylglycerol in potentiation of O~ production by using the DAG kinase inhibitor R 59022. Furthermore, a good correlation has been found, both in the kinetics and magnitude, between DAG concentrations and superoxide production in response to various stimuli. 2°,2~ According to our direct evidence of PKC translocation in the membranes of MENK activated cells as well as to indirect evidence obtained by using PKC inhibitor H7, we have concluded that PKC is involved in MENK-induced ©Pearson Professional Ltd 1996

O~ release. H7 diminished the stimulatory effect of MENK in the low (10q2-10 -1° M) as well as in the higher (10-8 M) concentration range. It should be pointed out that 10-8 M MENK, induced not only the highest level of DAG formation, but also increased PKC translocation in the membranes. It is, however, surprising that the stimulatory effect of MENK, that is not (10-12 M) or is very moderate (10-~ M) associated with DAG formation, could be abolished by H7. Alike, the effect of MENK on DAG formation is very strong as compared to modest functional responses. It is possible that this occurs via activation of other mediators which stimulate DAG formation but need not be functionally associated with superoxide anion release. In accordance to this we have recently demonstrated that MENK could change the pattern of ILl and TNF activityY Neuropeptides (1996) 30(2), 193-201

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A number of observations support the hypothesis that PKC plays a role in oxidase activation. Salamino et al23 have found in a large number of subjects a highly significant correlation between the kinase level and the oxidative-burst response. Twomey et al24 have confirmed the important role of PKC in the respiratory burst using 3 selective PKC inhibitors. Under the experimental conditions of this study, MENK very rapidly (20 s) increased the level of free cytosolic calcium. The maximum observed between 25 and 30 s rapidly declined to the baseline value by 50 s. Thus, MENK exhibited a different kinetics compared to fMLP used as a positive control. Furthermore, the calcium peak induced by fMLP occurred earlier than the peak induced by MENK, it declined very slowly, and did not reach the baseline level within 60 s. Literature data show that the elevation cytosolic calcium caused by fMLP lasts for 4-5 minY Various agonists which stimulate O 2 release might elicit different patterns of calcium Neuropeptides (1996) 30(2), 193-201

mobilization and phosphoinoside remodelling. For example, fMLP is a potent ligand in evoking a rapid mobilization of calcium from intracellular stores, as well as a rapid influx of calcium from the extracellular medium, while concanavalin A-triggered increase in cytosolic calcium accrues predominantly from extracellular sources. On the basis of our results, it seems that MENK stimulates O 2 release through activation of the DAG/PKC pathway. However, in individual donors tested and in lower MENK concentration, a clear correlation between the level of DAG and O 2 production was not always observed, i.e. in some cases the 0 2 release was stimulated although the DAG level was in the control range. This might imply the involvement of another second messenger system transducting the MENK effects. In our experiments MENK-induced 0 2 stimulation could be blocked by NDGA, which indicates the possibility of the lipooxygenase pathway involvement. The activation of that pathway in the MENK induced respiratory burst was © Pearson Professional Ltd 1996

Neutrophil signal transduction in MENK modulated 0 d release

d e m o n s t r a t e d b y N a g y et al. 26 Literature d a t a s u p p o r t t h e view t h a t v a r i o u s c o n c e n t r a t i o n s of an agent, t h r o u g h h i g h a n d low affinity receptors, can u s e different s e c o n d messengers. For example, low c o n c e n t r a t i o n s of M E N K m o d u l a t e m a c r o p h a g e function, t h r o u g h 8 receptors positively c o u p l e d to g u a n y l a t e cyclase, while in h i g h e r conc e n t r a t i o n s t h e a c t i o n i n c l u d e s a d e n y l a t e cyctase a n d does n o t involve 8 receptors. 2 Collectively, o u r d a t a s u g g e s t t h a t M E N K i n d u c e d O~ release is a s s o c i a t e d w i t h a DAG/PKC signal t r a n s d u c t i o n p a t h w a y a c c o m p a n i e d w i t h a n increase of cytosolic Ca ++. Opposite to that, t h e s u p p r e s s i o n of O ; release i n d u c e d b y t h e m a i n d e g r a d a t i n g p r o d u c t of M E N K (Tyr-Gly-Gly) was n o t a s s o c i a t e d w i t h t h e s a m e signal t r a n s d u c t i o n mechanisms.

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