FEMS Microbiology Immunology 105 (1992)239-248 © 1992 Federation of European Microbiological Societies 0920-8534/92/$05.00 Published by Elsevier
239
FEMSIM 00281
Sequential phospholipase activation in the stimulation of the neutrophil NADPH oxidase F i o n a W a t s o n , J o h n J. R o b i n s o n a n d S t e v e n W. E d w a r d s Department of Biochemistry, University of Licerpool, Licerpool, UK
Key words: Respiratory burst; Receptor; Oxidant; Chemiluminescence
1. S U M M A R Y Stimulation of human neutrophils with the chemotactic peptide fMet-Leu-Phe results in activation of a rapid, transient burst of oxidant secretion, which reaches a maximal rate by about 1 rain after stimulation. This phase of oxidant secretion is then followed by intracellular oxidant production, which is detected by iuminol chemiluminescence but not by assays such as cytochrome c reduction or scopoletin oxidation. The rapid phase of oxidant secretion requires increases in intracellular free Ca 2+ and phospholipase A 2 activity, but not the activities of phospholipase D or protein kinase C. In contrast, intracellular oxidant production requires the activities of phospholipase D and protein kinase C. A model is thus proposed suggesting the sequential activation of different phospholipases which activate oxidase molecules on the plasma membrane or else from the membranes of specific granules.
2. I N T R O D U C T I O N The importance of the N A D P H oxidase of neutrophiis in microbial killing is highlighted in Correspondence to: F. Watson, Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK.
patients with chronic granulomatous disease (CGD) who have impaired ability to generate reactive oxidants and an increased susceptibility to pathogenic infections [1-3]. Analysis of the molecular defect(s) responsible for CGD and experiments with cell-free oxidase activation systems [4-6] have led to the identification of a number of both membrane-bound and cytosolic oxidase factors that assemble into an active complex during cell stimulation. The membranebound components include an unusual cytochrome b with a mid-point potential low enough to reduce 0 2 to 0 2 [7-9] and a flavoprotein [10], whilst the cytosolic factors include p47-phox and p66-phox [11,12]. Other factors such as GTP and arachidonic acid also appear to be intimately involved in oxidase activation. Because the reactive oxidants generated via this oxidase may, if they are secreted from neutrophils, indiscriminately attack host tissues, the oxidase is normally dormant in resting cells but is rapidly activated upon the neutrophil recognising specific signals in its micro-environment. Thus, all known pathophysiological agents that stimulate oxidant production are recognised by specific receptors on the surface of the neutrophil, and receptor occupancy is linked to oxidase activation via a network of second messenger systems. The signal transduction systems that have been implicated in oxidase activation are shown in Table 1 and include the products of several phospholipases, Ca2+/calmodulin systems and protein ki-
240 Table 1 Intracellular signalling systems implicated in NADPH oxidase activation of human neutrophils Enzyme Phospholipase Phospholipase Phospholipase Protein kinase
Function? C A2 D C
IP3, Ca 2+, diacylglycerol Arachidonic acid Phosphatidic acid, diacylglycerol Phosphorylation (activation?) of oxidase components
nase C [2,13]. Precisely how these systems are co-ordinated during oxidase activation is unknown and unravelling these problems is complicated because much of the available data in the literature is apparently contradictory. For example, inhibitors of protein kinase C only affect activation by some agonists, activation can be dissociated from intracellular Ca 2+ increases and the cell-free activation system requires neither Ca 2+, ATP (which would be needed for phosphorylations) nor specific granules (see later). In addition to the complex structure of the oxidase and the multiplicity of potential signal transduction systems, membrane-bound oxidase components (cytochrome b and flavoprotein) have a dual location within the neutrophil. These components are found on the plasma membrane but the bulk of the cellular content ( > 90%) is found from the membranes of specific granules [14]. Furthermore, whilst these specific granules translocate to the plasma membrane during activation [15], this process occurs too slowly to explain oxidase activation via translocation and assembly of the oxidase molecules on these granules. The specific granules also contain receptors (e.g. for fMet-Leu-Phe and CR3) and so degranulation will result in increases in the number of receptors and oxidase components. It is known that cell activation (e.g. with fMet-Leu-Phe) or priming with cytokines (e.g. GM-CSF) results in this recruitment of specific granules [16,17]. Thus, up-regulation of receptors and oxidase molecules onto the plasma membrane will enhance the ability of neutrophils to respond to patho-physiological challenge. However, it has recently been
shown that surface receptor expression and oxidase activity is extremely Iow in unprimed bloodstream neutrophils, but rapidly increased during priming or indeed during purification by many commonly-used methods [18,19]. Thus, priming may not merely up-regulate neutrophil function but rather may switch the cell from 'non-responder' to 'responder' status. The signal transduction systems regulating the translocation of specific granules to the plasma membrane are unknown and it is likely that this process is required to sustain oxidant production on the plasma membrane as assembled oxidase complexes become inactivated [20]. In addition to the ability of neutrophils to generate oxidants within phagolysosomes during phagocytosis, oxidase activation can occur on the plasma membrane such that oxidants can be secreted from the cell [21-24]. Indeed, this phenomenon forms the basis of assays such as superoxide dismutase-inhibitable cytochrome c reduction [25] and scopoletin oxidation [26] because the probes cannot permeate the cell and consequently only measure external oxidants. Small, diffusible probes such as luminol detect both extra- and intracellular oxidant production, [21] and luminol chemiluminescence has shown that distinct phases of oxidant secretion and intracellular production occur upon activation by agonists such as fMet-Leu-Phe [27]. The past history of the neutrophil (i.e. prior exposure to activating or priming signals) will determine the number of oxidase molecules and receptors on the cell surface and hence will presumably determine the extent to which activated cells can secrete oxidants. Such secretion (rather than intracellular oxidase activity) is likely to result in host tissue damage during neutrophil infiltration and indeed we have previously shown that neutrophils isolated from the synovial fluid of patients with active rheumatoid arthritis exhibit features to indicate that they have secreted reactive oxidants within the joint [28,29]. It is the aim of this work to determine if extraand intracellular oxidant production is regulated by distinct signal transduction pathways and to determine the role of oxidase components on specific granules in these processes.
241 3. M A T E R I A L S AND M E T H O D S
3.1. Preparation of neutrophils Neutrophils were isolated from heparinised venous blood of healthy volunteers using Mono Poly-Resolving Medium (Flow Laboratories) as described previously [30]. After purification, neutrophils were washed and suspended in RPMI 1640 medium (Flow Laboratories) and their purity ( > 97%) and viability ( > 95%) assessed by Wright's staining and Trypan blue exclusion, respectively. Ceils were counted using a Fuchs-Rosenthal haemocytometer slide after a suitable dilution and used within 5 h of preparation.
3.2. Effects of inhibitors of signal transduction pathways The inhibitory effect of the protein kinase C inhibitor, staurosporine, was tested by incubating neutrophils with 100 nM of this inhibitor for 2 rain prior to stimulation. Extending this preincubation period did not alter the kinetics of subsequent stimulated oxidant production, and these conditions resulted in complete inhibition of the PMA-stimulated N A D P H oxidase activity [27]. The effects of chloracysine [31,32], a phospholipase A2 inhibitor, and butanol [33], a phospholipase D inhibitor, on neutrophil function were tested over a range of concentrations: the final concentrations used are as indicated in the text.
3.3. Quin-2-AM loading Neutrophils were suspended in a K r e b s / H e p e s buffer [30] at 1 × 108 cells/ml and incubated with gentle agitation at 37°C for 10 rain in the presence of either 5 0 0 / z M Quin-2-AM or 1% DMSO as a solvent control. Samples were then diluted ten-fold with pre-warmed buffer and incubated for a further 20 min, prior to centrifugation at 700 x g for 3 rain. Cell pellets were then washed three times in buffer and finally suspended at 2 X 10 7 cells/ml in RPMI 1640 medium.
3.4. Chemiluminescence Purified neutrophils were suspended in RPMI 1640 medium containing 10 /xM luminol [34] at 0.5-1 x 106 cells/ml in a total volume of 1 ml at 37°C. Chemiluminescence was then measured us-
ing either a single channel LKB 1250 luminometer, an LKB 1251 25 tube luminometer or else a Dynatech ML-1000 plate reader.
3.5. Superoxide and hydrogen peroxide production This was measured as described in [25] as the rate of superoxide dismutase-inhibitable cytochrome c reduction: suspensions (1 ml) contained 75 /xM cytochrome c, 0.5-1 x 106 cells and, where indicated, 30 /xg/ml superoxide dismutase, and the absorption increase at 550 nm was measured using either a Perkin-Elmer Lambda 5 spectrophotometer or else a Bio-Rad 3550 kinetic plate reader. H 2 0 z production was measured by following the rate of oxidation of scopoletin [26].
3.6. Receptor analyses The monoclonal antibodies used were Leu 15 ( C D l l b ) and a non-immune IgG control of the appropriate sub-type, both from Becton and Dickinson: a n t i - C D l l b recognises an epitope on the a-chain of the CR3 receptor. For immunostaining of isolated neutrophils, cells were suspended in P B S / I % B S A / 0 . 1 % sodium azide, pH 7.2 and receptor expression was measured using a standard indirect immunofluorescence technique using FITC-labelled goat-(anti-mouse) immunoglobulin as a second layer [17]: both 1st and 2nd layer antibodies were added at saturating concentrations. In all experiments non-immune mouse IgG of the appropriate isotype was included as class specific 1st layer controls. Stained ceils were fixed in 1% paraformaldehyde in PBS and analysed using a Becton and Dickinson FACS Analyser I and a Consort 30 computer and software. The modal fluorescence was proportional to the number of antigenic sites per cell for each individual antibody. Fluorescence distributions represent a total of 5000 gated events.
3. 7. Materials Luminol, cytochrome c, Quin-2-AM and fMetLeu-Phe were from Sigma, whilst staurosporine was from Boehringer Mannheim. Mono-Poly Resolving Medium and RPMI 1640 medium were from Flow Laboratories. All monoclonal antibodies were from Becton and Dickinson, whilst all
242
other biochemicals were from Sigma. All other reagents were of the highest purity available.
4. R E S U L T S 4.1. Kinetics of intra- and extracellular oxidant production Upon stimulation of neutrophils with 1 /xM fMet-Leu-Phe, luminol chemiluminescence increased rapidly reaching a maximal value within 1-2 min after stimulation (Fig. la): activity then declined, but rose to a more sustained rate 6-8 min after stimulation. In contrast, measurements of either O y secretion (using superoxide dismutase-inhibitable cytochrome c reduction) or H 2 0 2
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Time (min) Fig. 1. Schematic representation o f N A D P H oxidase activation in h u m a n neutrophils in response to fMet-Leu-Phe. (a) Shows a chemiluminescence trace obtained after stimulation of 5 x 105 neutrophils (incubated in the presence of 10 /~M luminol), with 1 /zM fMet-Leu-Phe. Extracellular oxidant scavengers are: superoxide dismutase, 1 / x g / m l ; catalase, 2 /.Lg/ml; methionine, 0.25 m g / m l . (b) Shows superoxide secretion measured by superoxide dismutase inhibitable cytochrome c reduction or H 2 0 2 production measured by oxidation of scopoletin, as described in MATERIALS AND METHODS.
production (using oxidation of scopoletin) were monophasic because these assays only measure secreted oxidants (Fig. lb). When luminol chemiluminescence was measured in the presence of a cocktail of extracellular scavengers (superoxide dismutase, catalase and methionine), the initial phase of oxidant activity was not observed (confirming its extracellular nature) whilst the later activity was largely unaffected (confirming its intracellular location). 4.2. Role of intracellular Ca 2 + Loading of cells with the fluorescent Ca 2+ indicator Quin-2-AM can be used to measure changes in intracellular Ca 2+ levels during cell activation [35]. However, because this compound has a relatively high affinity for Ca 2+, it can also act as an intracellular Ca 2+ buffer, if loaded at sufficiently high concentrations [36]. Thus, when fMet-Leu-Phe was added to neutrophil suspensions which were buffered from intracellular Ca 2 + changes, the initial, extracellular oxidase activity was not observed (Fig. 2a). However, intracellular oxidase activity was largely unaffected by this treatment. Similarly, in Quin-2 loaded cells no O~- secretion was observed after fMet-Leu-Phe stimulation. These data indicate that rises in intracellular Ca 2+ are necessary for activation of the rapid phase of oxidant secretion, but this second messenger plays a less important role in the later stages of oxidant production. 4.3. Role of phospholipase A 2 The importance of this enzyme in oxidase activation was determined using the inhibitor chloracysine [31,32]. Incubation of neutrophils with this inhibitor at a concentration of 0.1 mM completely prevented the initial phase of both luminol chemiluminescence (Fig. 3a) and 0 2 secretion (Fig. 3b). Additionally, the later stages of intracellular oxidant production were inhibited by over 60% when the activity of phospholipase A 2 was blocked. These data indicate that a product of phospholipase A 2 (presumably arachidonic acid) is essential for the rapid burst of reactive oxidant secre-
243
tion and also plays a major role in the processes which lead to intracellular oxidase activation.
a) Chemiluminescen~
lO 8
4.4. Role of phospholipase D
8
Control
6
The role of phospholipase D in neutrophil activation can be determined by exploiting the fact that this enzyme will transphosphatidylate primary alcohols (such as ethanol and butanol) at the expense of its normal substrate, phosphatidylcholine [33]. Thus, primary alcohols are competitive inhibitors of this enzyme. Addition of 10 m M butanol to neutrophil suspensions had little effect on either the initiation
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Time (rain) Fig. 2. Role of intracellular C a 2+ in activation of reactive oxidant production. Neutrophils were incubated in the presence and absence of 500 /zM Quin-2-AM as described in MATERIALSAND METHODS.(a) Ceils were incubated in the presence of 10/~M luminol prior to measurement of chemiluminescence whilst in (b) cells were incubated in 75 /zM cytochrome c prior to measurement of 02 secretion. Stimulation was achieved by the addition of 1 #M fMet-Leu-Phe.
2
4 Time (rnin)
6
, + 0.1 mM ch~oracysine 8
Fig. 3. Role of phospholipase A 2 in oxidase activation. Neutrophils were incubated as described in MATERIALSAND METHODS in the presence and absence of 0.1 mM chloracysine prior to measurement of luminol chemiluminescence (a) or 0 2 secretion (b), stimulated by the addition of 1 /zM fMet-LeuPhe.
or maximal rate of O~- secretion in fMet-leu-Phe activated neutrophils (Fig. 4a). However, in suspensions in which phospholipase D activity was blocked, these maximal rates of 0 2 secretion were not sustained for the same duration as in control cells. Furthermore, whilst extracellular luminol chemiluminescence was largely unaffected by this inhibitor, the later stages of oxidant production were greatly decreased (Fig. 4b). These observations indicate that phospholipase D activity has a negligible role in activation of the initial burst of reactive oxidant secretion. However, this enzyme is important in sustaining oxidant production either on the plasma m e m brane or else at the intracellular site(s).
4.5. Role of protein kinase C In neutrophil suspensions in which protein kinase C activity was inhibited by 100 nM stau-
244 possible that recruitment a n d / o r activation of oxidase molecules (cytochrome b and flavoprotein) from the membranes of the specific granules are needed for sustained or intracellular oxidant production. Thus, the translocation of specific granules to the plasma membrane was assessed by measuring the surface expression of C D l l b [17]. Stimulation of neutrophils for 15 min with 1 /~M fMet-Leu-Phe (Fig. 6a) or priming with GMCSF resulted in an increased expression of C D l l b on the plasma membrane due to recruitment of specific granules. The addition of 30 mM butanol completely inhibited this up-regulation (Fig. 6b). Interestingly, 0.1 mM chloracysine inhibited C D l l b up-regulation in response to GM-CSF (data not shown) but had little effect on translocation stimulated by fMet-Leu-Phe (fig. 6b). These observations indicate that products of phospholipase A 2 and D are important for the molecular events regulating sub-cellular re-distribution of specific granules. As these granule
rosporine [27], there was little effect on either the initiation or maximal rate of 0 2 secretion stimulated by the addition of fMet-Leu-Phe (Fig. 5a). However, as was observed in experiments performed in the presence of butanol, this maximal rate of secretion was not sustained when protein kinase C activity was inhibited. Similarly, staurosporine had little or no effect on either the initiation or maximal rate of extracellular luminol chemiluminescence, but did inhibit the later stages of oxidase activity (Fig. 5b). Thus, protein kinase C has a negligible role in activation of the initial burst of oxidase secretion. This enzyme does, however, play a role in sustaining oxidase activity and is important in regulating extracellular oxidant production.
4.6. Role of phospholipases D and A 2 in specific granule translocation The above data indicated that the rapid burst of oxidant secretion may arise from activation of oxidase molecules on the plasma membrane. It is
a) Cytochromec reduction
b] Luminolchemiluminescence
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Fig. 4. Role of phospholipase D in oxidase activation. Neutrophils were incubated in the presence or absence of 10 m M butanol prior to the m e a s u r e m e n t of (a) O~- secretion or (b) luminol chemiluminescence stimulated by the addition of 1/~M fMet-Leu-Phe.
245 are these systems temporally organised?; what is their role in activating the rapid phase of oxidant secretion and the sustained or intracellular oxidant production?; and which systems regulate oxidase activity on the plasma membrane and specific granules? We propose the following model which answers these questions and is based upon a sequential activation of these signalling systems (Fig. 7). Upon activation with fMet-Leu-Phe, there is a rapid initiation of reactive oxidant secretion which
membranes also contain cytochrome b and flavoprotein oxidase components, inhibition of these phospholipases will prevent the recruitment of these components.
5. D I S C U S S I O N These experiments set out to answer the following questions: what signal transduction systems regulate N A D P H oxidase activation?; how
a) Cytochrome c reduction (0; secretion)
b) Luminol chemiluminescence
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4 6 0 4 8 Time (rain) Time (min) Fig. 5. Role of protein kinase C in oxidase activation. Neutrophils were incubated in the presence or absence of 100 nM staurosporine prior to the measurement of (a) 0 2 secretion or (b) luminol chemiluminescencestimulated by the addition of 1 t~M fMet-Leu-Phe.
246
peaks by about 1 min and thereafter declines, presumably because assembled orddase complexes become inactivated. This activity results from activation of cytochrome b and flavoprotein molecules already present on the plasma membrane, activation requiring the translocation/ assembly of the cytosolic factors with these plasma membrane components. This process requires transients in intracellular free Ca 2+ and the activity of phospholipase A 2. Possibly the increased intracellular Ca 2+ concentration plays a role in activating this lipase, although in vitro evidence suggests that the Ca 2+ requirement for phospholipase A 2 activation is in excess of the (even localised) concentration likely to be attained in vivo. Perhaps G protein interaction or some other co-valent modification lowers the Ca 2+ activation threshold for this lipase. The major product of a)
fMet-Leu-Phe Stimulated Oxidase Activity
239
FEMSIM 00281
Sequential phospholipase activation in the stimulation of the neutrophil NADPH oxidase F i o n a W a t s o n , J o h n J. R o b i n s o n a n d S t e v e n W. E d w a r d s Department of Biochemistry, University of Licerpool, Licerpool, UK
Key words: Respiratory burst; Receptor; Oxidant; Chemiluminescence
1. S U M M A R Y Stimulation of human neutrophils with the chemotactic peptide fMet-Leu-Phe results in activation of a rapid, transient burst of oxidant secretion, which reaches a maximal rate by about 1 rain after stimulation. This phase of oxidant secretion is then followed by intracellular oxidant production, which is detected by iuminol chemiluminescence but not by assays such as cytochrome c reduction or scopoletin oxidation. The rapid phase of oxidant secretion requires increases in intracellular free Ca 2+ and phospholipase A 2 activity, but not the activities of phospholipase D or protein kinase C. In contrast, intracellular oxidant production requires the activities of phospholipase D and protein kinase C. A model is thus proposed suggesting the sequential activation of different phospholipases which activate oxidase molecules on the plasma membrane or else from the membranes of specific granules.
2. I N T R O D U C T I O N The importance of the N A D P H oxidase of neutrophiis in microbial killing is highlighted in Correspondence to: F. Watson, Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK.
patients with chronic granulomatous disease (CGD) who have impaired ability to generate reactive oxidants and an increased susceptibility to pathogenic infections [1-3]. Analysis of the molecular defect(s) responsible for CGD and experiments with cell-free oxidase activation systems [4-6] have led to the identification of a number of both membrane-bound and cytosolic oxidase factors that assemble into an active complex during cell stimulation. The membranebound components include an unusual cytochrome b with a mid-point potential low enough to reduce 0 2 to 0 2 [7-9] and a flavoprotein [10], whilst the cytosolic factors include p47-phox and p66-phox [11,12]. Other factors such as GTP and arachidonic acid also appear to be intimately involved in oxidase activation. Because the reactive oxidants generated via this oxidase may, if they are secreted from neutrophils, indiscriminately attack host tissues, the oxidase is normally dormant in resting cells but is rapidly activated upon the neutrophil recognising specific signals in its micro-environment. Thus, all known pathophysiological agents that stimulate oxidant production are recognised by specific receptors on the surface of the neutrophil, and receptor occupancy is linked to oxidase activation via a network of second messenger systems. The signal transduction systems that have been implicated in oxidase activation are shown in Table 1 and include the products of several phospholipases, Ca2+/calmodulin systems and protein ki-
240 Table 1 Intracellular signalling systems implicated in NADPH oxidase activation of human neutrophils Enzyme Phospholipase Phospholipase Phospholipase Protein kinase
Function? C A2 D C
IP3, Ca 2+, diacylglycerol Arachidonic acid Phosphatidic acid, diacylglycerol Phosphorylation (activation?) of oxidase components
nase C [2,13]. Precisely how these systems are co-ordinated during oxidase activation is unknown and unravelling these problems is complicated because much of the available data in the literature is apparently contradictory. For example, inhibitors of protein kinase C only affect activation by some agonists, activation can be dissociated from intracellular Ca 2+ increases and the cell-free activation system requires neither Ca 2+, ATP (which would be needed for phosphorylations) nor specific granules (see later). In addition to the complex structure of the oxidase and the multiplicity of potential signal transduction systems, membrane-bound oxidase components (cytochrome b and flavoprotein) have a dual location within the neutrophil. These components are found on the plasma membrane but the bulk of the cellular content ( > 90%) is found from the membranes of specific granules [14]. Furthermore, whilst these specific granules translocate to the plasma membrane during activation [15], this process occurs too slowly to explain oxidase activation via translocation and assembly of the oxidase molecules on these granules. The specific granules also contain receptors (e.g. for fMet-Leu-Phe and CR3) and so degranulation will result in increases in the number of receptors and oxidase components. It is known that cell activation (e.g. with fMet-Leu-Phe) or priming with cytokines (e.g. GM-CSF) results in this recruitment of specific granules [16,17]. Thus, up-regulation of receptors and oxidase molecules onto the plasma membrane will enhance the ability of neutrophils to respond to patho-physiological challenge. However, it has recently been
shown that surface receptor expression and oxidase activity is extremely Iow in unprimed bloodstream neutrophils, but rapidly increased during priming or indeed during purification by many commonly-used methods [18,19]. Thus, priming may not merely up-regulate neutrophil function but rather may switch the cell from 'non-responder' to 'responder' status. The signal transduction systems regulating the translocation of specific granules to the plasma membrane are unknown and it is likely that this process is required to sustain oxidant production on the plasma membrane as assembled oxidase complexes become inactivated [20]. In addition to the ability of neutrophils to generate oxidants within phagolysosomes during phagocytosis, oxidase activation can occur on the plasma membrane such that oxidants can be secreted from the cell [21-24]. Indeed, this phenomenon forms the basis of assays such as superoxide dismutase-inhibitable cytochrome c reduction [25] and scopoletin oxidation [26] because the probes cannot permeate the cell and consequently only measure external oxidants. Small, diffusible probes such as luminol detect both extra- and intracellular oxidant production, [21] and luminol chemiluminescence has shown that distinct phases of oxidant secretion and intracellular production occur upon activation by agonists such as fMet-Leu-Phe [27]. The past history of the neutrophil (i.e. prior exposure to activating or priming signals) will determine the number of oxidase molecules and receptors on the cell surface and hence will presumably determine the extent to which activated cells can secrete oxidants. Such secretion (rather than intracellular oxidase activity) is likely to result in host tissue damage during neutrophil infiltration and indeed we have previously shown that neutrophils isolated from the synovial fluid of patients with active rheumatoid arthritis exhibit features to indicate that they have secreted reactive oxidants within the joint [28,29]. It is the aim of this work to determine if extraand intracellular oxidant production is regulated by distinct signal transduction pathways and to determine the role of oxidase components on specific granules in these processes.
241 3. M A T E R I A L S AND M E T H O D S
3.1. Preparation of neutrophils Neutrophils were isolated from heparinised venous blood of healthy volunteers using Mono Poly-Resolving Medium (Flow Laboratories) as described previously [30]. After purification, neutrophils were washed and suspended in RPMI 1640 medium (Flow Laboratories) and their purity ( > 97%) and viability ( > 95%) assessed by Wright's staining and Trypan blue exclusion, respectively. Ceils were counted using a Fuchs-Rosenthal haemocytometer slide after a suitable dilution and used within 5 h of preparation.
3.2. Effects of inhibitors of signal transduction pathways The inhibitory effect of the protein kinase C inhibitor, staurosporine, was tested by incubating neutrophils with 100 nM of this inhibitor for 2 rain prior to stimulation. Extending this preincubation period did not alter the kinetics of subsequent stimulated oxidant production, and these conditions resulted in complete inhibition of the PMA-stimulated N A D P H oxidase activity [27]. The effects of chloracysine [31,32], a phospholipase A2 inhibitor, and butanol [33], a phospholipase D inhibitor, on neutrophil function were tested over a range of concentrations: the final concentrations used are as indicated in the text.
3.3. Quin-2-AM loading Neutrophils were suspended in a K r e b s / H e p e s buffer [30] at 1 × 108 cells/ml and incubated with gentle agitation at 37°C for 10 rain in the presence of either 5 0 0 / z M Quin-2-AM or 1% DMSO as a solvent control. Samples were then diluted ten-fold with pre-warmed buffer and incubated for a further 20 min, prior to centrifugation at 700 x g for 3 rain. Cell pellets were then washed three times in buffer and finally suspended at 2 X 10 7 cells/ml in RPMI 1640 medium.
3.4. Chemiluminescence Purified neutrophils were suspended in RPMI 1640 medium containing 10 /xM luminol [34] at 0.5-1 x 106 cells/ml in a total volume of 1 ml at 37°C. Chemiluminescence was then measured us-
ing either a single channel LKB 1250 luminometer, an LKB 1251 25 tube luminometer or else a Dynatech ML-1000 plate reader.
3.5. Superoxide and hydrogen peroxide production This was measured as described in [25] as the rate of superoxide dismutase-inhibitable cytochrome c reduction: suspensions (1 ml) contained 75 /xM cytochrome c, 0.5-1 x 106 cells and, where indicated, 30 /xg/ml superoxide dismutase, and the absorption increase at 550 nm was measured using either a Perkin-Elmer Lambda 5 spectrophotometer or else a Bio-Rad 3550 kinetic plate reader. H 2 0 z production was measured by following the rate of oxidation of scopoletin [26].
3.6. Receptor analyses The monoclonal antibodies used were Leu 15 ( C D l l b ) and a non-immune IgG control of the appropriate sub-type, both from Becton and Dickinson: a n t i - C D l l b recognises an epitope on the a-chain of the CR3 receptor. For immunostaining of isolated neutrophils, cells were suspended in P B S / I % B S A / 0 . 1 % sodium azide, pH 7.2 and receptor expression was measured using a standard indirect immunofluorescence technique using FITC-labelled goat-(anti-mouse) immunoglobulin as a second layer [17]: both 1st and 2nd layer antibodies were added at saturating concentrations. In all experiments non-immune mouse IgG of the appropriate isotype was included as class specific 1st layer controls. Stained ceils were fixed in 1% paraformaldehyde in PBS and analysed using a Becton and Dickinson FACS Analyser I and a Consort 30 computer and software. The modal fluorescence was proportional to the number of antigenic sites per cell for each individual antibody. Fluorescence distributions represent a total of 5000 gated events.
3. 7. Materials Luminol, cytochrome c, Quin-2-AM and fMetLeu-Phe were from Sigma, whilst staurosporine was from Boehringer Mannheim. Mono-Poly Resolving Medium and RPMI 1640 medium were from Flow Laboratories. All monoclonal antibodies were from Becton and Dickinson, whilst all
242
other biochemicals were from Sigma. All other reagents were of the highest purity available.
4. R E S U L T S 4.1. Kinetics of intra- and extracellular oxidant production Upon stimulation of neutrophils with 1 /xM fMet-Leu-Phe, luminol chemiluminescence increased rapidly reaching a maximal value within 1-2 min after stimulation (Fig. la): activity then declined, but rose to a more sustained rate 6-8 min after stimulation. In contrast, measurements of either O y secretion (using superoxide dismutase-inhibitable cytochrome c reduction) or H 2 0 2
Extracellular --
Intracellular I
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8
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oxidant scavengers
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10
Time (min) Fig. 1. Schematic representation o f N A D P H oxidase activation in h u m a n neutrophils in response to fMet-Leu-Phe. (a) Shows a chemiluminescence trace obtained after stimulation of 5 x 105 neutrophils (incubated in the presence of 10 /~M luminol), with 1 /zM fMet-Leu-Phe. Extracellular oxidant scavengers are: superoxide dismutase, 1 / x g / m l ; catalase, 2 /.Lg/ml; methionine, 0.25 m g / m l . (b) Shows superoxide secretion measured by superoxide dismutase inhibitable cytochrome c reduction or H 2 0 2 production measured by oxidation of scopoletin, as described in MATERIALS AND METHODS.
production (using oxidation of scopoletin) were monophasic because these assays only measure secreted oxidants (Fig. lb). When luminol chemiluminescence was measured in the presence of a cocktail of extracellular scavengers (superoxide dismutase, catalase and methionine), the initial phase of oxidant activity was not observed (confirming its extracellular nature) whilst the later activity was largely unaffected (confirming its intracellular location). 4.2. Role of intracellular Ca 2 + Loading of cells with the fluorescent Ca 2+ indicator Quin-2-AM can be used to measure changes in intracellular Ca 2+ levels during cell activation [35]. However, because this compound has a relatively high affinity for Ca 2+, it can also act as an intracellular Ca 2+ buffer, if loaded at sufficiently high concentrations [36]. Thus, when fMet-Leu-Phe was added to neutrophil suspensions which were buffered from intracellular Ca 2 + changes, the initial, extracellular oxidase activity was not observed (Fig. 2a). However, intracellular oxidase activity was largely unaffected by this treatment. Similarly, in Quin-2 loaded cells no O~- secretion was observed after fMet-Leu-Phe stimulation. These data indicate that rises in intracellular Ca 2+ are necessary for activation of the rapid phase of oxidant secretion, but this second messenger plays a less important role in the later stages of oxidant production. 4.3. Role of phospholipase A 2 The importance of this enzyme in oxidase activation was determined using the inhibitor chloracysine [31,32]. Incubation of neutrophils with this inhibitor at a concentration of 0.1 mM completely prevented the initial phase of both luminol chemiluminescence (Fig. 3a) and 0 2 secretion (Fig. 3b). Additionally, the later stages of intracellular oxidant production were inhibited by over 60% when the activity of phospholipase A 2 was blocked. These data indicate that a product of phospholipase A 2 (presumably arachidonic acid) is essential for the rapid burst of reactive oxidant secre-
243
tion and also plays a major role in the processes which lead to intracellular oxidase activation.
a) Chemiluminescen~
lO 8
4.4. Role of phospholipase D
8
Control
6
The role of phospholipase D in neutrophil activation can be determined by exploiting the fact that this enzyme will transphosphatidylate primary alcohols (such as ethanol and butanol) at the expense of its normal substrate, phosphatidylcholine [33]. Thus, primary alcohols are competitive inhibitors of this enzyme. Addition of 10 m M butanol to neutrophil suspensions had little effect on either the initiation
~
.E O
2
+ 0.1 mM chloracysine
0
2
4 "rime(rain)
6
b) Superoxidesecretion 0.8 0.6 ~
Chemdumlnescenee 8 Ia )
~
ox
Control
i
8
¢9
Control
0.4 0.2
c
0.0 0
E O 0
I
I
4
8
Time (min)
Superoxide secretion
92
%
t
0
1
2
3
4
5
6
Time (rain) Fig. 2. Role of intracellular C a 2+ in activation of reactive oxidant production. Neutrophils were incubated in the presence and absence of 500 /zM Quin-2-AM as described in MATERIALSAND METHODS.(a) Ceils were incubated in the presence of 10/~M luminol prior to measurement of chemiluminescence whilst in (b) cells were incubated in 75 /zM cytochrome c prior to measurement of 02 secretion. Stimulation was achieved by the addition of 1 #M fMet-Leu-Phe.
2
4 Time (rnin)
6
, + 0.1 mM ch~oracysine 8
Fig. 3. Role of phospholipase A 2 in oxidase activation. Neutrophils were incubated as described in MATERIALSAND METHODS in the presence and absence of 0.1 mM chloracysine prior to measurement of luminol chemiluminescence (a) or 0 2 secretion (b), stimulated by the addition of 1 /zM fMet-LeuPhe.
or maximal rate of O~- secretion in fMet-leu-Phe activated neutrophils (Fig. 4a). However, in suspensions in which phospholipase D activity was blocked, these maximal rates of 0 2 secretion were not sustained for the same duration as in control cells. Furthermore, whilst extracellular luminol chemiluminescence was largely unaffected by this inhibitor, the later stages of oxidant production were greatly decreased (Fig. 4b). These observations indicate that phospholipase D activity has a negligible role in activation of the initial burst of reactive oxidant secretion. However, this enzyme is important in sustaining oxidant production either on the plasma m e m brane or else at the intracellular site(s).
4.5. Role of protein kinase C In neutrophil suspensions in which protein kinase C activity was inhibited by 100 nM stau-
244 possible that recruitment a n d / o r activation of oxidase molecules (cytochrome b and flavoprotein) from the membranes of the specific granules are needed for sustained or intracellular oxidant production. Thus, the translocation of specific granules to the plasma membrane was assessed by measuring the surface expression of C D l l b [17]. Stimulation of neutrophils for 15 min with 1 /~M fMet-Leu-Phe (Fig. 6a) or priming with GMCSF resulted in an increased expression of C D l l b on the plasma membrane due to recruitment of specific granules. The addition of 30 mM butanol completely inhibited this up-regulation (Fig. 6b). Interestingly, 0.1 mM chloracysine inhibited C D l l b up-regulation in response to GM-CSF (data not shown) but had little effect on translocation stimulated by fMet-Leu-Phe (fig. 6b). These observations indicate that products of phospholipase A 2 and D are important for the molecular events regulating sub-cellular re-distribution of specific granules. As these granule
rosporine [27], there was little effect on either the initiation or maximal rate of 0 2 secretion stimulated by the addition of fMet-Leu-Phe (Fig. 5a). However, as was observed in experiments performed in the presence of butanol, this maximal rate of secretion was not sustained when protein kinase C activity was inhibited. Similarly, staurosporine had little or no effect on either the initiation or maximal rate of extracellular luminol chemiluminescence, but did inhibit the later stages of oxidase activity (Fig. 5b). Thus, protein kinase C has a negligible role in activation of the initial burst of oxidase secretion. This enzyme does, however, play a role in sustaining oxidase activity and is important in regulating extracellular oxidant production.
4.6. Role of phospholipases D and A 2 in specific granule translocation The above data indicated that the rapid burst of oxidant secretion may arise from activation of oxidase molecules on the plasma membrane. It is
a) Cytochromec reduction
b] Luminolchemiluminescence
(o; s ~
Control /~
10 mM Butanol
~I
0.025
L 0
1
I
2 Time (min)
1 3
0
4 Time (rain)
8
Fig. 4. Role of phospholipase D in oxidase activation. Neutrophils were incubated in the presence or absence of 10 m M butanol prior to the m e a s u r e m e n t of (a) O~- secretion or (b) luminol chemiluminescence stimulated by the addition of 1/~M fMet-Leu-Phe.
245 are these systems temporally organised?; what is their role in activating the rapid phase of oxidant secretion and the sustained or intracellular oxidant production?; and which systems regulate oxidase activity on the plasma membrane and specific granules? We propose the following model which answers these questions and is based upon a sequential activation of these signalling systems (Fig. 7). Upon activation with fMet-Leu-Phe, there is a rapid initiation of reactive oxidant secretion which
membranes also contain cytochrome b and flavoprotein oxidase components, inhibition of these phospholipases will prevent the recruitment of these components.
5. D I S C U S S I O N These experiments set out to answer the following questions: what signal transduction systems regulate N A D P H oxidase activation?; how
a) Cytochrome c reduction (0; secretion)
b) Luminol chemiluminescence
Control
Control
~porine 4 mV
Staurosporine
0.06
~' 0
I 2
I
I
V
I
J
4 6 0 4 8 Time (rain) Time (min) Fig. 5. Role of protein kinase C in oxidase activation. Neutrophils were incubated in the presence or absence of 100 nM staurosporine prior to the measurement of (a) 0 2 secretion or (b) luminol chemiluminescencestimulated by the addition of 1 t~M fMet-Leu-Phe.
246
peaks by about 1 min and thereafter declines, presumably because assembled orddase complexes become inactivated. This activity results from activation of cytochrome b and flavoprotein molecules already present on the plasma membrane, activation requiring the translocation/ assembly of the cytosolic factors with these plasma membrane components. This process requires transients in intracellular free Ca 2+ and the activity of phospholipase A 2. Possibly the increased intracellular Ca 2+ concentration plays a role in activating this lipase, although in vitro evidence suggests that the Ca 2+ requirement for phospholipase A 2 activation is in excess of the (even localised) concentration likely to be attained in vivo. Perhaps G protein interaction or some other co-valent modification lowers the Ca 2+ activation threshold for this lipase. The major product of a)
fMet-Leu-Phe Stimulated Oxidase Activity
