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Stimulation of Primed Neutrophils by Soluble Immune Complexes: Priming Leads to Enhanced Intracellular Ca2+Elevations, Activation of Phospholipase D, and Activation of the NADPH Oxidase
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
247, 819–826 (1998)
RC988524
Stimulation of Primed Neutrophils by Soluble Immune Complexes: Priming Leads to Enhanced Intracellular Ca2/ Elevations, Activation of Phospholipase D, and Activation of the NADPH Oxidase Fiona Watson1 and Steven W. Edwards2 School of Biological Sciences, Life Sciences Building, University of Liverpool, P.O. Box 147, Liverpool L69 7ZB, United Kingdom
Received February 27, 1998
Previous work has shown that synovial fluid from patients with rheumatoid arthritis contains two types
of IgG-containing immune complex (1-3) which have different molecular properties (4-6). The first type of complexes are insoluble and activate primed or unprimed neutrophils with near equal potency, but are primarily phagocytosed by the neutrophils. Thus, the bulk of the reactive oxidants are generated within phagolysosomes with few released extracellularly. The second type of immune complexes are soluble. These do not activate reactive oxidant production in unprimed neutrophils, but result in a rapid and transient release of oxidants and granule enzymes from neutrophils that have been primed either in vitro (e.g. with GM-CSF3 or g-interferon) or in vivo (4). We have also shown (7) that synthetic soluble and insoluble immune complexes (made in vitro from human serum albumin (HSA) and anti-HSA antibodies) activate neutrophils by analagous mechanisms to those complexes isolated from synovial fluid. Because neutrophils within rheumatoid joints have been primed in vivo (8,9) we have proposed that the soluble immune complexes are of great importance in disease pathology because they activate the secretion of large quantities of tissue-damaging products from infiltrating neutrophils. The aim of this work, therefore, was to identify the molecular changes that occur during priming which allow soluble immune complexes to activate secretion. Both synthetic and rheumatoid soluble complexes are IgG-containing and thus bind to the neutrophil cell surface via Fc receptors (4). Indeed, we have previously shown that activation of oxidant secretion in response to soluble complexes is blocked when ligation to either FcgRII or FcgRIII is experimentally manipulated (10).
1 Present address: The Physiology Laboratory, University of Liverpool, Liverpool L69 3BX. 2 All correspondence to: Dr. S. W. Edwards. Fax: 44 (0) 151 4349. E-mail: [email protected].
3 Abbreviations used: FITC, fluorescein isothiocyanate; IgG, immunoglobulin G; GM-CSF, granulocyte-macrophage colony-stimulating factor; HSA, human serum albumin; O20, superoxide; Hepes, N-[2hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid].
Soluble immune complexes activate a rapid burst of reactive oxidant secretion from neutrophils that have previously been primed with GM-CSF. Binding of these complexes to the cell surface of unprimed neutrophils results in the generation of intracellular Ca2/ transients, but the NADPH oxidase fails to become activated. No phospholipase D activity was observed following the addition of soluble immune complexes to unprimed cells. Upon priming with GM-CSF, the intracellular Ca2/ signal generated following soluble complex binding was greatly extended and phospholipase D was activated: there was also increased phosphorylation of proteins on tyrosine residues and the NADPH oxidase was activated. When Ca2/ influx was prevented, this phospholipase D activity was not observed. This primed oxidase activity was completely inhibited by erbstatin. Treatment of unprimed neutrophils with pervanadate (to inhibit protein tyrosine phosphatases) mimicked the effects of priming in that pervanadate-treated neutrophils secreted reactive oxidants in response to soluble immune complexes. The data indicate that during priming a new signaling pathway is activated that involves Ca2/ influx, phosphorylation on tyrosine residues, phospholipase D activity, and NADPH oxidase activation. q 1998 Academic Press
Key Words: receptors; respiratory burst; rheumatoid arthritis; tyrosine kinase; GM-CSF.
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However, priming has little effect on the overall surface level of expression of either FcgRII or FcgRIII (11,12), and so it seems unlikely that changes in Fcg receptor expression alone are a sufficient explanation for the ability of soluble complexes to stimulate secretion from primed neutrophils. Here, we show that the intracellular signalling systems activated by soluble immune complexes are dramatically altered during priming. Soluble immune complexes bind to the surface of unprimed neutrophils and receptor binding results in the generation of intracellular Ca2/ transients, but these are insufficient to activate the oxidase: phospholipase D is not activated under these conditions. After priming, the intracellular Ca2/ signal is extended via Ca2/ influx and phospholipase D becomes activated. In parallel with these changes, there are alterations in phosphorylation on tyrosine residues and such phosphorylations appear to be intimately linked with the priming process. MATERIALS AND METHODS Materials. Mono-Poly Resolving Medium, RPMI 1640 medium were from Flow laboratories (Paisley, Scotland), human serum albumin (HSA), anti-HSA antibodies, FITC, luminol, cytochalasin B, superoxide dismutase, protease inhibitors and cytochrome c were from Sigma. Fluo-3-AM, genestein and erbstatin were from Calbiochem, 3 H-lysoPAF and the ECL detection kit were from Amersham (U.K.). Antibody PY20 was from ICN Biochemicals. GM-CSF was a nonglycosylated protein from Glaxo. Isolation of neutrophils. Neutrophils were isolated from the venous blood of healthy volunteers by centrifugation in Mono-Poly Resolving Medium as described previously (13). After removal of contaminating erythrocytes by hypotonic lysis, purified neutrophils were suspended in RPMI 1640 medium and counted (after a suitable dilution) using a Fuchs-Rosenthal hemocytometer. Neutrophil purity (assessed by Wright’s staining) and viability (assessed by trypan blue exclusion) were routinely measured and found to be ú97% and ú95 %, respectively. Neutrophils were used within 4 h of preparation. Priming was achieved by incubation with rGM-CSF (50 U/ml) for 1 h at 377C. Preparation of synthetic immune complexes. Synthetic immune complexes were made using human serum albumin (HSA) and rabbit anti-(human HSA) antibodies as described in (14). The zone of equivalence (the optimal antigen:antibody ratio required for the formation of insoluble complexes) was determined by adding HSA and anti(HSA) antibodies and measuring the absorption at 450 nm. Soluble immune complexes were formed at six-fold the concentration of antigen required to form insoluble complexes. After formation, soluble immune complexes were briefly centrifuged (2 min at 13000 g in a microfuge) to remove any contaminating insoluble complexes that may have been present. Soluble complexes were formed at 180 mg/ ml antigen and 125 mg of antibody and 100 ml were added to 1 ml neutrophil suspensions. Measurements of reactive oxidant production. For measurements of chemiluminescence, neutrophils were suspended at 5 1 105 cells/ ml in RPMI 1640 medium that was supplemented with 10 mM luminol. Suspensions were stimulated by the addition of soluble immune complexes and photon emission was followed at 377C in an LKB 1251 luminometer (15). For measurements of O0 2 secretion, neutrophils were suspended at 5 1 105 cells/ml in RPMI 1640 medium that was
supplemented with 75 mM cytochrome c (5,16). The total volume was 200 ml and the suspension was placed in 96 well microtitre plates. After addition of soluble immune complexes, absorption increases at 550 nm were recorded using a Bio-Rad 3550 kinetic plate reader. Reference wells also contained 30 mg/ml superoxide dismutase (SOD) and absorption values in these wells were subtracted to obtain SODinhibitable O20 secretion. Measurements of cytosolic free Ca2/. Neutrophil suspensions (2 1 107 cells/ ml in RPMI 1640 medium) were incubated for 30 min at 377C with 2 mM Fluo-3AM. The cells were then washed twice and suspended at 2 1 106 cells/ml in Ca2/ free medium (NaCl, 145 mM; Na2HPO4.2H2O, 1 mM; MgSO4.7H2O, 0.5 mM; glucose, 5 mM, Hepes, 20 mM, pH 7.4). Extracellular Ca2/ was added, as indicated as 1 mM CaCl2, whereas in some experiments no Ca2/ was added and experiments were performed in media containing 1 mM EGTA. Fluorescence was then measured at 505 nm excitation and 530 nm emission in 3 ml cuvettes using a Perkin-Elmer 3000 Fluorimeter after appropriate stimulation. Calibration of changes in intracellular Ca2/ levels was as described in (17) using a Kd for Fluo-3 of 864 nM Ca2/ at 377C. Measurement of phospholipase D activity. Labelling of neutrophils with [3H]alkyl-lysoPAF (5 mCi/ml), extraction of phospholipids and analysis of phospholipase D activity (phosphatidylethanol production) was performed as described previously (18,19). Measurement of tyrosine phosphorylation using immunoblotting. Following stimulation, 107 cells were pelleted at 47C and then resuspended in 0.5 ml lysis buffer containing 1% Triton X-100, 150 mM NaCl , 10 mM Tris pH 7.2, 5 mM EDTA, 13 mM sodium pyrophosphate, 50 mM NaF, 1.1 mM sodium orthovanadate, 1 mg/ml fatty acid free BSA, 1 mM PMSF, 3 mg/ml each of aprotinin, leupeptin, antipain, pepstatin A and chymostatin. The Triton insoluble fraction was then pelleted and 50 ml of 21 SDS sample buffer added to 150 ml of supernatant. The samples were electrophoresed on SDS-PAGE gels and then blotted onto Immobilon P membrane (Millipore). The membranes were blocked by incubation for 1h at 377C with a solution of 3 % gelatin in T/N TBS buffer (20 mM Tris pH 7.2, 100 mM NaCl, 0.05 % Nonidet P40, 0.05 % Tween 20). The membrane was washed briefly in T/N TBS buffer prior to a 2h incubation at room temperature in T/N TBS buffer containing 0.05 % gelatin and 0.075 mg/ml of the horseradish peroxidase linked anti-phosphotyrosine antibody PY20. The membrane was then washed 5 times in T/N TBS buffer, the first wash was of 15 min duration followed by 4 others of 5 min each. The membrane was then developed using ECL reagents from Amersham. Inhibition of tyrosine kinases and phosphatases. In order to inhibit tyrosine kinase activity prior to measurement of NADPH oxidase activity, neutrophils (5 1 105 cells/ml) were incubated for 5 min at room temperature with 1 mg/ml of erbstatin analogue. To inhibit tyrosine phosphatase activity, neutrophils (5 1 105 cells/ml) were incubated in the presence of 500 mM sodium pervanadate.
RESULTS Activation of reactive oxidant production by soluble immune complexes. When freshly-isolated blood neutrophils were incubated with soluble HSA/anti-(HSA) immune complexes, they could not activate the NADPH oxidase as indicated by a failure to generate luminol chemiluminescence (Fig. 1A) and inability to reduce cytochrome c (Fig 1B). However, when neutrophils were primed with GM-CSF prior to addition of soluble immune complexes, a remarkable change in their responsiveness was observed. In primed cells, the soluble
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FIG. 1. Stimulation of reactive oxidant production by soluble immune complexes. Neutrophils (107/ml) were isolated from the blood of healthy volunteers and suspended in RPMI 1640 medium in the absence (s) or presence (l) of 50 U/ml GM-CSF for 1 h at 377C. After incubation, 5 1 105 cells were incubated with synthetic soluble immune complexes (125 mg antibody:180 mg antigen) and the reactive oxidants produced were measured by either chemiluminescence (A) or cytochrome c reduction (B). For chemiluminescence measurements, suspensions (1 ml) were supplemented with 10 mM luminol, whilst in B suspensions contained 75 mM cytochrome c in a total volume of 200 ml and absorbance measurements were made using a kinetic plate reader: reference wells contained the same additions plus 30 mg/ml superoxide dismutase. Both assays were performed at 377C and similar results have been found in 10 separate experiments.
immune complexes activated a rapid, transient burst of chemiluminescence (Fig.1A). This activity reached a maximal rate 3 min after addition of stimulus and then declined to basal levels. Similarly, the soluble immune complexes activated a transient burst of O20 secretion as indicated by a marked increase in SOD-inhibitable cytochrome c reduction (Fig. 1B). Thus, a fundamental change in the molecular properties of neutrophils occurs during priming that allows the soluble complexes to activate the NADPH oxidase. The following experiments were thus designed to identify this crucial biochemical process. Activation of phospholipase C by soluble immune complexes. Changes in intracellular Ca2/ were followed after loading neutrophils with Fluo-3AM and measuring increases in fluorescence after addition of complexes. After loading, basal levels of fluorescence indicated that the resting level of intracellular Ca2/ in unstimulated cells was 139 nM ({ 45 nM, n Å 16), in agreement with previous reports (17). Addition of soluble immune complexes to unprimed cells resulted in the generation of a transient increase in intracellular Ca2/ (Fig. 2A). After addition of complexes there was a lag of approx. 20 sec before the intracellular Ca2/ levels began to increase. Levels reached 800-1000 nM by 45 sec and then declined to unstimulated levels. Priming of neutrophils with GM-CSF did not affect the basal intracellular Ca2/ level in unstimulated cells (Fig. 2A). Upon addition of soluble immune complexes to primed cells, both the lag phase and time to peak increase were virtually identical to those observed in
unprimed cells. However, in primed cells, the elevation in intracellular Ca2/ was more sustained than that observed in unprimed cells. Thus, in primed neutrophils, the soluble immune complexes stimulate the generation of an additional intracellular Ca2/ transient. When experiments were performed in Ca2/ free media (containing 1 mM EGTA) the unprimed intracellular Ca2/ transient was largely unaffected (Fig. 2B). However, the ‘‘extra’’ intracellular Ca2/ signal seen upon priming with GM-CSF was not seen (Fig. 2C). Thus, in unprimed neutrophils, the intracellular Ca2/ transient is largely due to mobilisation of intracellular stores, whereas the ‘‘extra’’ Ca2/ signal seen in primed cells is largely due to Ca2/ influx. These experiments indicate that in unprimed cells, because addition of soluble immune complexes activates increases in intracellular Ca2/, these complexes must bind to functional receptor(s). However, these Ca2/ transients are insufficient to activate the NADPH oxidase. Activation of phospholipase D by soluble immune complexes. The activity of phospholipase D was measured by formation of phosphatidylethanol in primed and unprimed cells. In unprimed neutrophils, soluble immune complexes activated only low levels of phosphatidylethanol production (Fig. 3A). However, in primed cells, higher levels of phosphatidylethanol were released, peaking 2 min after addition of complexes (Fig. 3A). In order to determine if the ‘‘extra’’ Ca2/ signal was involved in activation of phospholipase D, phosphatidylethanol production was measured in cultures stimu-
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FIG. 2. Elevations in intracellular Ca2/. Neutrophils were loaded with Fluo-3AM as described in Materials and Methods. In A, neutrophils were incubated in the presence and absence of GM-CSF (50 U/ml, for 30 min) prior to Fluo-3AM loading (for 30 min) followed by addition of soluble immune complexes In B and C, unprimed neutrophils and GM-CSF primed neutrophils, respectively, were incubated in either in Ca2/ containing medium (1 mM) or Ca2/ free medium containing 1 mM EGTA prior to addition of soluble immune complexes. Similar results were obtained in at least 7 other experiments.
lated with soluble immune complexes in the presence and absence of extracellular Ca2/. Figure 3B shows that in Ca2/ free media phospholipase D activity in GM-CSF primed was decreased to the unprimed level. Hence, when Ca2/ influx is prevented, the enhanced phospholipase D activity is not seen. It was then necessary to establish the role of extracellular Ca2/ in activation of the NADPH oxidase. The GM-CSF primed oxidase activity following addition of soluble immune complexes was inhibited by approx 50 % (52 % { 15 %, n Å 4) when suspensions were incubated in Ca2/ free medium (Fig. 3C). Thus, this priming dependent NADPH oxidase activity is only partly dependent upon Ca2/ influx. These experiments indicate that in unprimed cells, the soluble immune complexes activate phospholipase C, but activate only low levels of phospholipase D. This latter phospholipase is activated at high levels when the cells are primed, and thus it is likely that the products of this enzyme (phosphatidic acid/diacylglycerol) play a role in activation of the NADPH oxidase in primed cells. It is now clear that tyrosine kinase/tyrosine phosphatase activities are involved in the regulation of cell activation, and so the role of these processes in activation of the NADPH oxidase in primed and unprimed cells was investigated. Role of tyrosine kinases/tyrosine phosphatases in primed and unprimed neutrophils. In order to deter-
mine the role of phosphorylation of proteins on tyrosine residues during priming of the NADPH oxidase, 3 approaches were taken. Firstly, tyrosine phosphatase activity was inhibited by pervanadate. Secondly, tyrosine kinase activity was inhibited by erbstatin. Thirdly, anti-phosphotyrosine antibodies were used to identify phosphorylated proteins in western blots. Addition of soluble immune complexes to unprimed neutrophils failed to activate a respiratory burst, but when unprimed cells were pre-treated with pervanadate (to inhibit tyrosine phosphatases), addition of soluble immune complexes resulted in activation of the NADPH oxidase (Fig. 4A). Thus, inhibition of tyrosine phosphatases (leading to enhanced phosphorylation of proteins on tyrosine residues) mimics the priming effects of GM-CSF. The role of tyrosine kinase activity was further investigated using the inhibitor, erbstatin. Neutrophils were primed with GM-CSF and then incubated in the presence and absence of erbstatin prior to the addition of soluble immune complexes. In the absence of erbstatin the GM-CSF-primed cells generated a rapid transient burst of reactive oxidants (Fig. 4B). However, when tyrosine kinase activity was blocked, no reactive oxidants were generated. Erbstatin also inhibited the generation of O20 in primed cells, as assessed by the cytochrome c reduction assay (data not shown). Similar results were obtained using the inhibitor, genestein
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GM-CSF (Fig. 5) and stimulated with soluble immune complexes. Addition of GM-CSF alone to neutrophils slightly altered the profile of protein tyrosine phosphorylation. However, following addition of GM-CSF, the soluble immune complexes stimulated enhanced phosphorylation of proteins of around 21-22, 31-32, 33-34,
FIG. 3. Changes in phospholipase D activity following stimulation with soluble immune complexes. In A, neutrophils were preloaded with 3H-lyso-PAF, and then incubated with 100 mM ethanol after incubation in the absence (s) and presence (l) of 50 U/ml GMCSF, as described in Materials and Methods. After addition of soluble immune complexes (arrow), samples were removed for analysis of phosphatidylethanol by TLC. Similar results were obtained in 4 other experiments. In B, 3H-lyso-PAF loaded neutrophils were incubated with 100 mM ethanol and samples removed 2 min after addition of soluble immune complexes for analysis of phosphatidylethanol production. 1, unprimed cells; 2, cells incubated with 50 U/ml GMCSF prior to addition of complexes; 3, GM-CSF primed cells incubated in Ca2/ free medium containing 1 mM EGTA prior to addition of complexes. Error bars are standard deviations of 3-5 separate experiments. In C, neutrophils were primed with GM-CSF and then incubated in either Ca2/ containing medium (1) or Ca2/ free medium containing 1 mM EGTA (2). Luminol chemiluminescence was then measured after the addition of soluble immune complexes.
(data not shown). These data thus confirm the role of tyrosine kinase activity in the processes leading to oxidase activation in the response of primed cells to soluble immune complexes. The kinetics of phosphorylation of proteins on tyrosine residues were then directly followed by western blotting using anti-phosphotyrosine antibodies. Neutrophils were incubated in the presence and absence of
FIG. 4. Role of tyrosine kinases in neutrophil priming. In A, neutrophils were incubated for 5 min at 377C in the absence (l) or presence (s,h) of 500 mM pervanadate prior to addition of soluble immune complexes (s,l) and measurement of luminol chemiluminescence. In B, neutrophils were primed by incubation with 50 U/ ml GM-CSF and then incubated in the absence (l) and presence (s) of 1 mg/ml erbstatin analogue prior to addition of soluble immune complexes and measurement of luminol chemiluminescence.
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FIG. 5. Soluble immune complex-stimulated protein tyrosine phosphorylation. Neutrophils were incubated for 1 h in the presence and absence of 50 U/ml GM-CSF prior to addition of soluble immune complexes. At 30 sec, 2.5 min and 10 min after stimulation, samples were removed for analysis of tyrosine phosphoproteins. U, unprimed; GM, GM-CSF primed only; US, unprimed cells / soluble immune complexes; GMS, GM-CSF primed cells / soluble immune complexes. Bar markers (right hand margin) indicate molecular mass standards. Typical results of 3-5 separate experiments.
72-74 and 90 kDa. The soluble immune complexes stimulated phosphorylation of proteins of around 38-39, 4042, 61-62 and 63-64 kDa in both primed and unprimed cells. DISCUSSION We have shown that the soluble immune complexes interact with the neutrophil plasma membrane via the Fcg receptors, FcgRII and FcgRIII (10). However, surface levels of expression of these two receptors changes very little upon priming (11,12) and so it may have been predicted that the ability of primed neutrophils to respond to soluble immune complexes was not merely associated with alterations in binding to the cell surface. The Fcg receptor(s) bound by these complexes in unprimed cells are partially functional because binding resulted in the generation of intracellular Ca2/ transients. However, these elevations in intracellular Ca2/ are in themselves, insufficient to activate the oxidase, a situation that has been implied from other studies (20). When the neutrophils were primed, the soluble complexes resulted in a prolonged intracellular Ca2/ signal. This sustained increase in intracellular Ca2/ in primed cells appears to be due to enhanced Ca2/ influx from extracellular sources. It was then necessary to search for other signalling systems whose activity may be altered during priming. Much evidence in the literature implicates the role of phospholipases D in activation of the NADPH oxidase, via the generation of phosphatidic acid and diac-
ylglycerol (19-26). Phosphatidic acid can be converted into diacylglycerol by the activity of phosphatidate phosphohydrolase. This is believed to be the major route for the production of diacylglycerol in neutrophils, and is the physiological activator of protein kinase C. There is also some evidence to implicate phosphatidic acid itself as a signalling molecule involved in NADPH oxidase activation (27,28). There is also evidence in the literature to indicate that phospholipase D activity is enhanced in primed cells (19, 29,30). In unprimed cells, the soluble immune complexes could activate only low levels of phospholipase D, as indicated by low levels of formation of phosphatidylethanol. However, when the cells were primed, the soluble complexes could activate higher levels of phospholipase D, and the kinetics of formation closely coincided with activation of the oxidase. In view of the accepted importance of phosphatidic acid/diacylglycerol in activation of the NADPH oxidase, it is suggested that their increased production in primed cells plays a major role in the secretion of reactive oxidants following addition of soluble complexes. This enhanced phospholipase D activity was not seen in cells incubated in the absence of extracellular Ca2/. Thus, the ‘‘extra’’ Ca2/ signal seen in GM-CSF primed appears to play a role in phospholipase D activation. However, the primed NADPH oxiase activity was only decreased by about 50 % in supensions incubated in Ca2/ free conditions. This implies that the phospholipase D route accounts for about 50 % of the primed oxidase response. A second intracellular route for NADPH oxidase activation, that is indepen-
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dent of Ca2/ influx and phospholipase D activation must also exist. There is now much evidence in the literature implicating phosphorylation of proteins on tyrosine residues in the regulation of neutrophil function (31-33). We therefore investigated the possibility that changes in tyrosine phosphorylation levels could play a role in oxidase activation in primed cells. We first incubated neutrophils with pervanadate in the absence of an exogenous priming agent. Pervanadate is an inhibitor of protein tyrosine phosphatases (31) and so its addition to cells results in an increased level of phosphorylation on tyrosine residues via endogenous tyrosine kinase activity. When soluble immune complexes were added to pervanadate treated neutrophils, the NADPH oxidase could be activated. Thus, inhibition of tyrosine phosphatases and subsequent increased phosphorylation on tyrosine residues mimicked the effects of priming. This strongly implies a role for phosphorylation on tyrosine residues of protein(s) involved in the regulation of NADPH oxidase activation during priming. This suggestion was further supported by the observation that NADPH oxidase activity in primed cells was completely sensitive to inhibition by erbstatin, a potent inhibitor of protein tyrosine kinases. Furthermore, we were able to detect changes in labelling of tyrosine phosphoproteins following stimulation of primed and unprimed cells with soluble immune complexes. The time courses of these changes in phosphorylation patterns closely coincided with the kinetics of oxidase activity, strengthening the likelihood of their direct involvement. Further work is clearly necessary to identify these phosphoproteins and to establish their role in oxidase activation. It is possible that the 40 kDa phosphoprotein is FcgRII, which is known to undergo tyrosine phosphorylation (34,35). We therefore propose the following events to explain the interactions of soluble immune complexes with primed and unprimed neutrophils. In unprimed cells, the soluble complexes interact with FcgR and this receptor occupancy activates phospholipase C to transiently raise intracellular Ca2/ levels: these events do not result in activation of the oxidase and there is little activation of phospholipase D. When the cells are primed, the soluble immune complexes interact with the same receptors whose surface level of expression is largely unchanged during the priming process. However, this receptor-ligand interaction now results in increased Ca2/ influx and the enhanced activation of phospholipase D to generate phosphatidic acid. Because EGTA completely abolished the ‘‘extra’’ intracellular Ca2/ signal and this enhanced phospholipase D activity, the increased Ca2/ influx probably plays a role in activation of phospholipase D. However, EGTA treatment only partly blocked NADPH oxidase activity in primed cells and so an alternative, phospholipase D-
independent mechanism for oxidase activation must exist in primed cells. The increased levels of phosphorylation of proteins on tyrosine residues could be the result of increased activities of tyrosine kinases or decreased activity of tyrosine phosphatases, or a combination of the two. It is thus likely that increased levels of phosphorylation of key protein(s) on tyrosine residues provides the coupling link between occupancy of Fcg receptors and activation of phospholipase D, and that this is altered during priming. Further work is thus required to establish this hypothesis, to identify the key phosphoproteins and to establish whether analogous events also occur during priming of inflammatory neutrophils within rheumatoid joints. This could then lead to new ways to regulate the function of inflammatory neutrophils and hence decrease their damaging effects during inflammatory diseases. ACKNOWLEDGMENTS We thank the Arthritis and Rheumatism Council for financial support.
REFERENCES 1. Blackburn, W. D. J., Koopman, W. J., Schrohenloher, R. E., and Heck, L. W. (1986) Clin. Immunol. Immunopathol. 40, 347–355. 2. Bender, J. G., Van Epps, D. E., Searles, R., and Williams, R. C. J. (1986) Inflammation 10, 443–453. 3. Gale, R., Bertouch, J. V., Bradley, J., and Roberts-Thomson, P. J. (1983) Ann. Rheum. Dis. 42, 158–162. 4. Robinson, J. J., Watson, F., Bucknall, R. C., and Edwards, S. W. (1992) Biochem. J. 286, 345–351. 5. Robinson, J. J., Watson, F., Bucknall, R. C., and Edwards S. W. (1992) Eur. J. Clin. Invest. 22, 314–318. 6. Robinson, J. J., Watson, F., Phelan, M., Bucknall, R. C., and Edwards, S. W. (1993) Ann. Rheum. Dis. 52, 347–353. 7. Robinson, J. J., Watson, F., Bucknall, R. C., and Edwards, S. W. (1994) FEMS. Immunol. Med. Microbiol. 8, 249–258. 8. Nurcombe, H. L., Bucknall, R. C., and Edwards, S. W. (1991) Ann. Rheum. Dis. 51, 147–153 9. Dularay, B., Elson, C. J., and Dieppe, P. A. (1988) Autoimmunity 1, 159–169. 10. Robinson, J. J., Watson, F., Bucknall, R. C., and Edwards, S. W. (1994) Ann. Rheum. Dis. 53, 515–520. 11. Watson, F., Robinson, J. J., Phelan, M., Bucknall, R. C., and Edwards, S. W. (1993) Ann. Rheum. Dis. 52, 354–359. 12. Edwards, S. W., Watson, F., Macleod, R., and Davies, J. M. (1990) Biosci. Rep 10, 393–401. 13. Edwards, S. W., Say, J. E., and Hart, C. A. (1987) J. Gen. Microbiol. 133, 3591–3597 14. Crockett-Torabi, E., and Fantone, J. C. (1990) J. Immunol. 145, 3026–3032. 15. Edwards, S. W. (1987) J. Clin. Lab. Immunol. 22, 35–39. 16. Babior, B. M., Kipnes, R. S., and Curnutte, J. T. (1973) J. Clin. Invest. 52, 741–744. 17. Merritt, J. E., McCarthy, S. A., Davies, M. P. A., and Moores, K. E. (1990) Biochem. J. 269, 513–519.
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18. Lowe, G. M., Slupsky, J. R., Galvani, D. W., and Edwards, S. W. (1996) Biochem. Biophys. Res. Commun. 220, 484–490. 19. Watson, F., Lowe, G. M., Robinson, J. J., Galvani, D. W., and Edwards, S. W. (1994) Biosci. Rep. 14, 91–102. 20. Grinstein, S., and Furuya, W. (1988) J. Biol. Chem. 263, 1779– 1783. 21. Cockcroft, S. (1992) Biochem. Biophys. Acta 1113, 135–160. 22. Thompson, N. T., Tateson, J. E., Randall, R. W., Spacey, G. D., Bonser, R. W., and Garland, L. G. (1990) Biochem. J. 271, 209– 213. 23. Billah, M. M., Eckel, S., Mullmann, T. J., Egan, R. W., and Siegel, M. I. (1989) J. Biol. Chem. 264, 17069–17077. 24. Billah, M. M. (1993) Curr. Opin. Immunol. 5, 114–123. 25. Bonser, R. W., Thompson, N. T., Randall, R. W., and Garland, L. G. (1989) Biochem. J. 264, 617–620. 26. Whatmore, J., Cronin, P., and Cockcroft, S. (1994) FEBS Letts. 352, 113–117.
27. Agwu, D. E., McPhail, L. C., Sozzani, S., Bass, D. A., and McCall, C. E. (1991) J. Clin. Invest . 88, 531–539. 28. Rossi, F., Greskowiak, M., Della Blanca, V., Calzetti, F., and Gandini, G. (1990) Biochem. Biophys. Res. Commun. 168, 320– 327. 29. Bourgoin, S., Borgeat, P., and Poubelle, P. E. (1991) Agents Actions 34, 32–34. 30. Bourgoin, S., Poubelle, P. E., Liao, N. W., Umezawa, K., Borgeat, P., and Naccache, P. H. (1992) Cell. Signalling 4, 487–500. 31. Uings, I. J., Thompson, N. T., Randall, R. W., Spacey, G. D., Bonser, R. W., Hudson, A. T., and Garland, L. G. (1992) Biochem. J . 281, 597–600. 32. Berkow, R. L., and Dodson, R. W. (1990) Blood 75, 2445–2452. 33. Lloyds, D., and Hallett, M. B. (1994) Biochem. Pharmacol. 48, 15–21. 34. Dusi, S., Domini, M., Dellabianca, V., Gandini, G., and Rossi, F. (1994) Biochem. Biophys. Res. Commun. 201, 30–37. 35. Hamada, F., Aoki, M., Akiyama, T., and Toyoshima, K. (1993) Proc. Natn. Acad. Sci. USA 90, 6305–6309.
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Stimulation of Primed Neutrophils by Soluble Immune Complexes: Priming Leads to Enhanced Intracellular Ca2/ Elevations, Activation of Phospholipase D, and Activation of the NADPH Oxidase Fiona Watson1 and Steven W. Edwards2 School of Biological Sciences, Life Sciences Building, University of Liverpool, P.O. Box 147, Liverpool L69 7ZB, United Kingdom
Received February 27, 1998
Previous work has shown that synovial fluid from patients with rheumatoid arthritis contains two types
of IgG-containing immune complex (1-3) which have different molecular properties (4-6). The first type of complexes are insoluble and activate primed or unprimed neutrophils with near equal potency, but are primarily phagocytosed by the neutrophils. Thus, the bulk of the reactive oxidants are generated within phagolysosomes with few released extracellularly. The second type of immune complexes are soluble. These do not activate reactive oxidant production in unprimed neutrophils, but result in a rapid and transient release of oxidants and granule enzymes from neutrophils that have been primed either in vitro (e.g. with GM-CSF3 or g-interferon) or in vivo (4). We have also shown (7) that synthetic soluble and insoluble immune complexes (made in vitro from human serum albumin (HSA) and anti-HSA antibodies) activate neutrophils by analagous mechanisms to those complexes isolated from synovial fluid. Because neutrophils within rheumatoid joints have been primed in vivo (8,9) we have proposed that the soluble immune complexes are of great importance in disease pathology because they activate the secretion of large quantities of tissue-damaging products from infiltrating neutrophils. The aim of this work, therefore, was to identify the molecular changes that occur during priming which allow soluble immune complexes to activate secretion. Both synthetic and rheumatoid soluble complexes are IgG-containing and thus bind to the neutrophil cell surface via Fc receptors (4). Indeed, we have previously shown that activation of oxidant secretion in response to soluble complexes is blocked when ligation to either FcgRII or FcgRIII is experimentally manipulated (10).
1 Present address: The Physiology Laboratory, University of Liverpool, Liverpool L69 3BX. 2 All correspondence to: Dr. S. W. Edwards. Fax: 44 (0) 151 4349. E-mail: [email protected].
3 Abbreviations used: FITC, fluorescein isothiocyanate; IgG, immunoglobulin G; GM-CSF, granulocyte-macrophage colony-stimulating factor; HSA, human serum albumin; O20, superoxide; Hepes, N-[2hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid].
Soluble immune complexes activate a rapid burst of reactive oxidant secretion from neutrophils that have previously been primed with GM-CSF. Binding of these complexes to the cell surface of unprimed neutrophils results in the generation of intracellular Ca2/ transients, but the NADPH oxidase fails to become activated. No phospholipase D activity was observed following the addition of soluble immune complexes to unprimed cells. Upon priming with GM-CSF, the intracellular Ca2/ signal generated following soluble complex binding was greatly extended and phospholipase D was activated: there was also increased phosphorylation of proteins on tyrosine residues and the NADPH oxidase was activated. When Ca2/ influx was prevented, this phospholipase D activity was not observed. This primed oxidase activity was completely inhibited by erbstatin. Treatment of unprimed neutrophils with pervanadate (to inhibit protein tyrosine phosphatases) mimicked the effects of priming in that pervanadate-treated neutrophils secreted reactive oxidants in response to soluble immune complexes. The data indicate that during priming a new signaling pathway is activated that involves Ca2/ influx, phosphorylation on tyrosine residues, phospholipase D activity, and NADPH oxidase activation. q 1998 Academic Press
Key Words: receptors; respiratory burst; rheumatoid arthritis; tyrosine kinase; GM-CSF.
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However, priming has little effect on the overall surface level of expression of either FcgRII or FcgRIII (11,12), and so it seems unlikely that changes in Fcg receptor expression alone are a sufficient explanation for the ability of soluble complexes to stimulate secretion from primed neutrophils. Here, we show that the intracellular signalling systems activated by soluble immune complexes are dramatically altered during priming. Soluble immune complexes bind to the surface of unprimed neutrophils and receptor binding results in the generation of intracellular Ca2/ transients, but these are insufficient to activate the oxidase: phospholipase D is not activated under these conditions. After priming, the intracellular Ca2/ signal is extended via Ca2/ influx and phospholipase D becomes activated. In parallel with these changes, there are alterations in phosphorylation on tyrosine residues and such phosphorylations appear to be intimately linked with the priming process. MATERIALS AND METHODS Materials. Mono-Poly Resolving Medium, RPMI 1640 medium were from Flow laboratories (Paisley, Scotland), human serum albumin (HSA), anti-HSA antibodies, FITC, luminol, cytochalasin B, superoxide dismutase, protease inhibitors and cytochrome c were from Sigma. Fluo-3-AM, genestein and erbstatin were from Calbiochem, 3 H-lysoPAF and the ECL detection kit were from Amersham (U.K.). Antibody PY20 was from ICN Biochemicals. GM-CSF was a nonglycosylated protein from Glaxo. Isolation of neutrophils. Neutrophils were isolated from the venous blood of healthy volunteers by centrifugation in Mono-Poly Resolving Medium as described previously (13). After removal of contaminating erythrocytes by hypotonic lysis, purified neutrophils were suspended in RPMI 1640 medium and counted (after a suitable dilution) using a Fuchs-Rosenthal hemocytometer. Neutrophil purity (assessed by Wright’s staining) and viability (assessed by trypan blue exclusion) were routinely measured and found to be ú97% and ú95 %, respectively. Neutrophils were used within 4 h of preparation. Priming was achieved by incubation with rGM-CSF (50 U/ml) for 1 h at 377C. Preparation of synthetic immune complexes. Synthetic immune complexes were made using human serum albumin (HSA) and rabbit anti-(human HSA) antibodies as described in (14). The zone of equivalence (the optimal antigen:antibody ratio required for the formation of insoluble complexes) was determined by adding HSA and anti(HSA) antibodies and measuring the absorption at 450 nm. Soluble immune complexes were formed at six-fold the concentration of antigen required to form insoluble complexes. After formation, soluble immune complexes were briefly centrifuged (2 min at 13000 g in a microfuge) to remove any contaminating insoluble complexes that may have been present. Soluble complexes were formed at 180 mg/ ml antigen and 125 mg of antibody and 100 ml were added to 1 ml neutrophil suspensions. Measurements of reactive oxidant production. For measurements of chemiluminescence, neutrophils were suspended at 5 1 105 cells/ ml in RPMI 1640 medium that was supplemented with 10 mM luminol. Suspensions were stimulated by the addition of soluble immune complexes and photon emission was followed at 377C in an LKB 1251 luminometer (15). For measurements of O0 2 secretion, neutrophils were suspended at 5 1 105 cells/ml in RPMI 1640 medium that was
supplemented with 75 mM cytochrome c (5,16). The total volume was 200 ml and the suspension was placed in 96 well microtitre plates. After addition of soluble immune complexes, absorption increases at 550 nm were recorded using a Bio-Rad 3550 kinetic plate reader. Reference wells also contained 30 mg/ml superoxide dismutase (SOD) and absorption values in these wells were subtracted to obtain SODinhibitable O20 secretion. Measurements of cytosolic free Ca2/. Neutrophil suspensions (2 1 107 cells/ ml in RPMI 1640 medium) were incubated for 30 min at 377C with 2 mM Fluo-3AM. The cells were then washed twice and suspended at 2 1 106 cells/ml in Ca2/ free medium (NaCl, 145 mM; Na2HPO4.2H2O, 1 mM; MgSO4.7H2O, 0.5 mM; glucose, 5 mM, Hepes, 20 mM, pH 7.4). Extracellular Ca2/ was added, as indicated as 1 mM CaCl2, whereas in some experiments no Ca2/ was added and experiments were performed in media containing 1 mM EGTA. Fluorescence was then measured at 505 nm excitation and 530 nm emission in 3 ml cuvettes using a Perkin-Elmer 3000 Fluorimeter after appropriate stimulation. Calibration of changes in intracellular Ca2/ levels was as described in (17) using a Kd for Fluo-3 of 864 nM Ca2/ at 377C. Measurement of phospholipase D activity. Labelling of neutrophils with [3H]alkyl-lysoPAF (5 mCi/ml), extraction of phospholipids and analysis of phospholipase D activity (phosphatidylethanol production) was performed as described previously (18,19). Measurement of tyrosine phosphorylation using immunoblotting. Following stimulation, 107 cells were pelleted at 47C and then resuspended in 0.5 ml lysis buffer containing 1% Triton X-100, 150 mM NaCl , 10 mM Tris pH 7.2, 5 mM EDTA, 13 mM sodium pyrophosphate, 50 mM NaF, 1.1 mM sodium orthovanadate, 1 mg/ml fatty acid free BSA, 1 mM PMSF, 3 mg/ml each of aprotinin, leupeptin, antipain, pepstatin A and chymostatin. The Triton insoluble fraction was then pelleted and 50 ml of 21 SDS sample buffer added to 150 ml of supernatant. The samples were electrophoresed on SDS-PAGE gels and then blotted onto Immobilon P membrane (Millipore). The membranes were blocked by incubation for 1h at 377C with a solution of 3 % gelatin in T/N TBS buffer (20 mM Tris pH 7.2, 100 mM NaCl, 0.05 % Nonidet P40, 0.05 % Tween 20). The membrane was washed briefly in T/N TBS buffer prior to a 2h incubation at room temperature in T/N TBS buffer containing 0.05 % gelatin and 0.075 mg/ml of the horseradish peroxidase linked anti-phosphotyrosine antibody PY20. The membrane was then washed 5 times in T/N TBS buffer, the first wash was of 15 min duration followed by 4 others of 5 min each. The membrane was then developed using ECL reagents from Amersham. Inhibition of tyrosine kinases and phosphatases. In order to inhibit tyrosine kinase activity prior to measurement of NADPH oxidase activity, neutrophils (5 1 105 cells/ml) were incubated for 5 min at room temperature with 1 mg/ml of erbstatin analogue. To inhibit tyrosine phosphatase activity, neutrophils (5 1 105 cells/ml) were incubated in the presence of 500 mM sodium pervanadate.
RESULTS Activation of reactive oxidant production by soluble immune complexes. When freshly-isolated blood neutrophils were incubated with soluble HSA/anti-(HSA) immune complexes, they could not activate the NADPH oxidase as indicated by a failure to generate luminol chemiluminescence (Fig. 1A) and inability to reduce cytochrome c (Fig 1B). However, when neutrophils were primed with GM-CSF prior to addition of soluble immune complexes, a remarkable change in their responsiveness was observed. In primed cells, the soluble
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FIG. 1. Stimulation of reactive oxidant production by soluble immune complexes. Neutrophils (107/ml) were isolated from the blood of healthy volunteers and suspended in RPMI 1640 medium in the absence (s) or presence (l) of 50 U/ml GM-CSF for 1 h at 377C. After incubation, 5 1 105 cells were incubated with synthetic soluble immune complexes (125 mg antibody:180 mg antigen) and the reactive oxidants produced were measured by either chemiluminescence (A) or cytochrome c reduction (B). For chemiluminescence measurements, suspensions (1 ml) were supplemented with 10 mM luminol, whilst in B suspensions contained 75 mM cytochrome c in a total volume of 200 ml and absorbance measurements were made using a kinetic plate reader: reference wells contained the same additions plus 30 mg/ml superoxide dismutase. Both assays were performed at 377C and similar results have been found in 10 separate experiments.
immune complexes activated a rapid, transient burst of chemiluminescence (Fig.1A). This activity reached a maximal rate 3 min after addition of stimulus and then declined to basal levels. Similarly, the soluble immune complexes activated a transient burst of O20 secretion as indicated by a marked increase in SOD-inhibitable cytochrome c reduction (Fig. 1B). Thus, a fundamental change in the molecular properties of neutrophils occurs during priming that allows the soluble complexes to activate the NADPH oxidase. The following experiments were thus designed to identify this crucial biochemical process. Activation of phospholipase C by soluble immune complexes. Changes in intracellular Ca2/ were followed after loading neutrophils with Fluo-3AM and measuring increases in fluorescence after addition of complexes. After loading, basal levels of fluorescence indicated that the resting level of intracellular Ca2/ in unstimulated cells was 139 nM ({ 45 nM, n Å 16), in agreement with previous reports (17). Addition of soluble immune complexes to unprimed cells resulted in the generation of a transient increase in intracellular Ca2/ (Fig. 2A). After addition of complexes there was a lag of approx. 20 sec before the intracellular Ca2/ levels began to increase. Levels reached 800-1000 nM by 45 sec and then declined to unstimulated levels. Priming of neutrophils with GM-CSF did not affect the basal intracellular Ca2/ level in unstimulated cells (Fig. 2A). Upon addition of soluble immune complexes to primed cells, both the lag phase and time to peak increase were virtually identical to those observed in
unprimed cells. However, in primed cells, the elevation in intracellular Ca2/ was more sustained than that observed in unprimed cells. Thus, in primed neutrophils, the soluble immune complexes stimulate the generation of an additional intracellular Ca2/ transient. When experiments were performed in Ca2/ free media (containing 1 mM EGTA) the unprimed intracellular Ca2/ transient was largely unaffected (Fig. 2B). However, the ‘‘extra’’ intracellular Ca2/ signal seen upon priming with GM-CSF was not seen (Fig. 2C). Thus, in unprimed neutrophils, the intracellular Ca2/ transient is largely due to mobilisation of intracellular stores, whereas the ‘‘extra’’ Ca2/ signal seen in primed cells is largely due to Ca2/ influx. These experiments indicate that in unprimed cells, because addition of soluble immune complexes activates increases in intracellular Ca2/, these complexes must bind to functional receptor(s). However, these Ca2/ transients are insufficient to activate the NADPH oxidase. Activation of phospholipase D by soluble immune complexes. The activity of phospholipase D was measured by formation of phosphatidylethanol in primed and unprimed cells. In unprimed neutrophils, soluble immune complexes activated only low levels of phosphatidylethanol production (Fig. 3A). However, in primed cells, higher levels of phosphatidylethanol were released, peaking 2 min after addition of complexes (Fig. 3A). In order to determine if the ‘‘extra’’ Ca2/ signal was involved in activation of phospholipase D, phosphatidylethanol production was measured in cultures stimu-
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FIG. 2. Elevations in intracellular Ca2/. Neutrophils were loaded with Fluo-3AM as described in Materials and Methods. In A, neutrophils were incubated in the presence and absence of GM-CSF (50 U/ml, for 30 min) prior to Fluo-3AM loading (for 30 min) followed by addition of soluble immune complexes In B and C, unprimed neutrophils and GM-CSF primed neutrophils, respectively, were incubated in either in Ca2/ containing medium (1 mM) or Ca2/ free medium containing 1 mM EGTA prior to addition of soluble immune complexes. Similar results were obtained in at least 7 other experiments.
lated with soluble immune complexes in the presence and absence of extracellular Ca2/. Figure 3B shows that in Ca2/ free media phospholipase D activity in GM-CSF primed was decreased to the unprimed level. Hence, when Ca2/ influx is prevented, the enhanced phospholipase D activity is not seen. It was then necessary to establish the role of extracellular Ca2/ in activation of the NADPH oxidase. The GM-CSF primed oxidase activity following addition of soluble immune complexes was inhibited by approx 50 % (52 % { 15 %, n Å 4) when suspensions were incubated in Ca2/ free medium (Fig. 3C). Thus, this priming dependent NADPH oxidase activity is only partly dependent upon Ca2/ influx. These experiments indicate that in unprimed cells, the soluble immune complexes activate phospholipase C, but activate only low levels of phospholipase D. This latter phospholipase is activated at high levels when the cells are primed, and thus it is likely that the products of this enzyme (phosphatidic acid/diacylglycerol) play a role in activation of the NADPH oxidase in primed cells. It is now clear that tyrosine kinase/tyrosine phosphatase activities are involved in the regulation of cell activation, and so the role of these processes in activation of the NADPH oxidase in primed and unprimed cells was investigated. Role of tyrosine kinases/tyrosine phosphatases in primed and unprimed neutrophils. In order to deter-
mine the role of phosphorylation of proteins on tyrosine residues during priming of the NADPH oxidase, 3 approaches were taken. Firstly, tyrosine phosphatase activity was inhibited by pervanadate. Secondly, tyrosine kinase activity was inhibited by erbstatin. Thirdly, anti-phosphotyrosine antibodies were used to identify phosphorylated proteins in western blots. Addition of soluble immune complexes to unprimed neutrophils failed to activate a respiratory burst, but when unprimed cells were pre-treated with pervanadate (to inhibit tyrosine phosphatases), addition of soluble immune complexes resulted in activation of the NADPH oxidase (Fig. 4A). Thus, inhibition of tyrosine phosphatases (leading to enhanced phosphorylation of proteins on tyrosine residues) mimics the priming effects of GM-CSF. The role of tyrosine kinase activity was further investigated using the inhibitor, erbstatin. Neutrophils were primed with GM-CSF and then incubated in the presence and absence of erbstatin prior to the addition of soluble immune complexes. In the absence of erbstatin the GM-CSF-primed cells generated a rapid transient burst of reactive oxidants (Fig. 4B). However, when tyrosine kinase activity was blocked, no reactive oxidants were generated. Erbstatin also inhibited the generation of O20 in primed cells, as assessed by the cytochrome c reduction assay (data not shown). Similar results were obtained using the inhibitor, genestein
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GM-CSF (Fig. 5) and stimulated with soluble immune complexes. Addition of GM-CSF alone to neutrophils slightly altered the profile of protein tyrosine phosphorylation. However, following addition of GM-CSF, the soluble immune complexes stimulated enhanced phosphorylation of proteins of around 21-22, 31-32, 33-34,
FIG. 3. Changes in phospholipase D activity following stimulation with soluble immune complexes. In A, neutrophils were preloaded with 3H-lyso-PAF, and then incubated with 100 mM ethanol after incubation in the absence (s) and presence (l) of 50 U/ml GMCSF, as described in Materials and Methods. After addition of soluble immune complexes (arrow), samples were removed for analysis of phosphatidylethanol by TLC. Similar results were obtained in 4 other experiments. In B, 3H-lyso-PAF loaded neutrophils were incubated with 100 mM ethanol and samples removed 2 min after addition of soluble immune complexes for analysis of phosphatidylethanol production. 1, unprimed cells; 2, cells incubated with 50 U/ml GMCSF prior to addition of complexes; 3, GM-CSF primed cells incubated in Ca2/ free medium containing 1 mM EGTA prior to addition of complexes. Error bars are standard deviations of 3-5 separate experiments. In C, neutrophils were primed with GM-CSF and then incubated in either Ca2/ containing medium (1) or Ca2/ free medium containing 1 mM EGTA (2). Luminol chemiluminescence was then measured after the addition of soluble immune complexes.
(data not shown). These data thus confirm the role of tyrosine kinase activity in the processes leading to oxidase activation in the response of primed cells to soluble immune complexes. The kinetics of phosphorylation of proteins on tyrosine residues were then directly followed by western blotting using anti-phosphotyrosine antibodies. Neutrophils were incubated in the presence and absence of
FIG. 4. Role of tyrosine kinases in neutrophil priming. In A, neutrophils were incubated for 5 min at 377C in the absence (l) or presence (s,h) of 500 mM pervanadate prior to addition of soluble immune complexes (s,l) and measurement of luminol chemiluminescence. In B, neutrophils were primed by incubation with 50 U/ ml GM-CSF and then incubated in the absence (l) and presence (s) of 1 mg/ml erbstatin analogue prior to addition of soluble immune complexes and measurement of luminol chemiluminescence.
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FIG. 5. Soluble immune complex-stimulated protein tyrosine phosphorylation. Neutrophils were incubated for 1 h in the presence and absence of 50 U/ml GM-CSF prior to addition of soluble immune complexes. At 30 sec, 2.5 min and 10 min after stimulation, samples were removed for analysis of tyrosine phosphoproteins. U, unprimed; GM, GM-CSF primed only; US, unprimed cells / soluble immune complexes; GMS, GM-CSF primed cells / soluble immune complexes. Bar markers (right hand margin) indicate molecular mass standards. Typical results of 3-5 separate experiments.
72-74 and 90 kDa. The soluble immune complexes stimulated phosphorylation of proteins of around 38-39, 4042, 61-62 and 63-64 kDa in both primed and unprimed cells. DISCUSSION We have shown that the soluble immune complexes interact with the neutrophil plasma membrane via the Fcg receptors, FcgRII and FcgRIII (10). However, surface levels of expression of these two receptors changes very little upon priming (11,12) and so it may have been predicted that the ability of primed neutrophils to respond to soluble immune complexes was not merely associated with alterations in binding to the cell surface. The Fcg receptor(s) bound by these complexes in unprimed cells are partially functional because binding resulted in the generation of intracellular Ca2/ transients. However, these elevations in intracellular Ca2/ are in themselves, insufficient to activate the oxidase, a situation that has been implied from other studies (20). When the neutrophils were primed, the soluble complexes resulted in a prolonged intracellular Ca2/ signal. This sustained increase in intracellular Ca2/ in primed cells appears to be due to enhanced Ca2/ influx from extracellular sources. It was then necessary to search for other signalling systems whose activity may be altered during priming. Much evidence in the literature implicates the role of phospholipases D in activation of the NADPH oxidase, via the generation of phosphatidic acid and diac-
ylglycerol (19-26). Phosphatidic acid can be converted into diacylglycerol by the activity of phosphatidate phosphohydrolase. This is believed to be the major route for the production of diacylglycerol in neutrophils, and is the physiological activator of protein kinase C. There is also some evidence to implicate phosphatidic acid itself as a signalling molecule involved in NADPH oxidase activation (27,28). There is also evidence in the literature to indicate that phospholipase D activity is enhanced in primed cells (19, 29,30). In unprimed cells, the soluble immune complexes could activate only low levels of phospholipase D, as indicated by low levels of formation of phosphatidylethanol. However, when the cells were primed, the soluble complexes could activate higher levels of phospholipase D, and the kinetics of formation closely coincided with activation of the oxidase. In view of the accepted importance of phosphatidic acid/diacylglycerol in activation of the NADPH oxidase, it is suggested that their increased production in primed cells plays a major role in the secretion of reactive oxidants following addition of soluble complexes. This enhanced phospholipase D activity was not seen in cells incubated in the absence of extracellular Ca2/. Thus, the ‘‘extra’’ Ca2/ signal seen in GM-CSF primed appears to play a role in phospholipase D activation. However, the primed NADPH oxiase activity was only decreased by about 50 % in supensions incubated in Ca2/ free conditions. This implies that the phospholipase D route accounts for about 50 % of the primed oxidase response. A second intracellular route for NADPH oxidase activation, that is indepen-
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dent of Ca2/ influx and phospholipase D activation must also exist. There is now much evidence in the literature implicating phosphorylation of proteins on tyrosine residues in the regulation of neutrophil function (31-33). We therefore investigated the possibility that changes in tyrosine phosphorylation levels could play a role in oxidase activation in primed cells. We first incubated neutrophils with pervanadate in the absence of an exogenous priming agent. Pervanadate is an inhibitor of protein tyrosine phosphatases (31) and so its addition to cells results in an increased level of phosphorylation on tyrosine residues via endogenous tyrosine kinase activity. When soluble immune complexes were added to pervanadate treated neutrophils, the NADPH oxidase could be activated. Thus, inhibition of tyrosine phosphatases and subsequent increased phosphorylation on tyrosine residues mimicked the effects of priming. This strongly implies a role for phosphorylation on tyrosine residues of protein(s) involved in the regulation of NADPH oxidase activation during priming. This suggestion was further supported by the observation that NADPH oxidase activity in primed cells was completely sensitive to inhibition by erbstatin, a potent inhibitor of protein tyrosine kinases. Furthermore, we were able to detect changes in labelling of tyrosine phosphoproteins following stimulation of primed and unprimed cells with soluble immune complexes. The time courses of these changes in phosphorylation patterns closely coincided with the kinetics of oxidase activity, strengthening the likelihood of their direct involvement. Further work is clearly necessary to identify these phosphoproteins and to establish their role in oxidase activation. It is possible that the 40 kDa phosphoprotein is FcgRII, which is known to undergo tyrosine phosphorylation (34,35). We therefore propose the following events to explain the interactions of soluble immune complexes with primed and unprimed neutrophils. In unprimed cells, the soluble complexes interact with FcgR and this receptor occupancy activates phospholipase C to transiently raise intracellular Ca2/ levels: these events do not result in activation of the oxidase and there is little activation of phospholipase D. When the cells are primed, the soluble immune complexes interact with the same receptors whose surface level of expression is largely unchanged during the priming process. However, this receptor-ligand interaction now results in increased Ca2/ influx and the enhanced activation of phospholipase D to generate phosphatidic acid. Because EGTA completely abolished the ‘‘extra’’ intracellular Ca2/ signal and this enhanced phospholipase D activity, the increased Ca2/ influx probably plays a role in activation of phospholipase D. However, EGTA treatment only partly blocked NADPH oxidase activity in primed cells and so an alternative, phospholipase D-
independent mechanism for oxidase activation must exist in primed cells. The increased levels of phosphorylation of proteins on tyrosine residues could be the result of increased activities of tyrosine kinases or decreased activity of tyrosine phosphatases, or a combination of the two. It is thus likely that increased levels of phosphorylation of key protein(s) on tyrosine residues provides the coupling link between occupancy of Fcg receptors and activation of phospholipase D, and that this is altered during priming. Further work is thus required to establish this hypothesis, to identify the key phosphoproteins and to establish whether analogous events also occur during priming of inflammatory neutrophils within rheumatoid joints. This could then lead to new ways to regulate the function of inflammatory neutrophils and hence decrease their damaging effects during inflammatory diseases. ACKNOWLEDGMENTS We thank the Arthritis and Rheumatism Council for financial support.
REFERENCES 1. Blackburn, W. D. J., Koopman, W. J., Schrohenloher, R. E., and Heck, L. W. (1986) Clin. Immunol. Immunopathol. 40, 347–355. 2. Bender, J. G., Van Epps, D. E., Searles, R., and Williams, R. C. J. (1986) Inflammation 10, 443–453. 3. Gale, R., Bertouch, J. V., Bradley, J., and Roberts-Thomson, P. J. (1983) Ann. Rheum. Dis. 42, 158–162. 4. Robinson, J. J., Watson, F., Bucknall, R. C., and Edwards, S. W. (1992) Biochem. J. 286, 345–351. 5. Robinson, J. J., Watson, F., Bucknall, R. C., and Edwards S. W. (1992) Eur. J. Clin. Invest. 22, 314–318. 6. Robinson, J. J., Watson, F., Phelan, M., Bucknall, R. C., and Edwards, S. W. (1993) Ann. Rheum. Dis. 52, 347–353. 7. Robinson, J. J., Watson, F., Bucknall, R. C., and Edwards, S. W. (1994) FEMS. Immunol. Med. Microbiol. 8, 249–258. 8. Nurcombe, H. L., Bucknall, R. C., and Edwards, S. W. (1991) Ann. Rheum. Dis. 51, 147–153 9. Dularay, B., Elson, C. J., and Dieppe, P. A. (1988) Autoimmunity 1, 159–169. 10. Robinson, J. J., Watson, F., Bucknall, R. C., and Edwards, S. W. (1994) Ann. Rheum. Dis. 53, 515–520. 11. Watson, F., Robinson, J. J., Phelan, M., Bucknall, R. C., and Edwards, S. W. (1993) Ann. Rheum. Dis. 52, 354–359. 12. Edwards, S. W., Watson, F., Macleod, R., and Davies, J. M. (1990) Biosci. Rep 10, 393–401. 13. Edwards, S. W., Say, J. E., and Hart, C. A. (1987) J. Gen. Microbiol. 133, 3591–3597 14. Crockett-Torabi, E., and Fantone, J. C. (1990) J. Immunol. 145, 3026–3032. 15. Edwards, S. W. (1987) J. Clin. Lab. Immunol. 22, 35–39. 16. Babior, B. M., Kipnes, R. S., and Curnutte, J. T. (1973) J. Clin. Invest. 52, 741–744. 17. Merritt, J. E., McCarthy, S. A., Davies, M. P. A., and Moores, K. E. (1990) Biochem. J. 269, 513–519.
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18. Lowe, G. M., Slupsky, J. R., Galvani, D. W., and Edwards, S. W. (1996) Biochem. Biophys. Res. Commun. 220, 484–490. 19. Watson, F., Lowe, G. M., Robinson, J. J., Galvani, D. W., and Edwards, S. W. (1994) Biosci. Rep. 14, 91–102. 20. Grinstein, S., and Furuya, W. (1988) J. Biol. Chem. 263, 1779– 1783. 21. Cockcroft, S. (1992) Biochem. Biophys. Acta 1113, 135–160. 22. Thompson, N. T., Tateson, J. E., Randall, R. W., Spacey, G. D., Bonser, R. W., and Garland, L. G. (1990) Biochem. J. 271, 209– 213. 23. Billah, M. M., Eckel, S., Mullmann, T. J., Egan, R. W., and Siegel, M. I. (1989) J. Biol. Chem. 264, 17069–17077. 24. Billah, M. M. (1993) Curr. Opin. Immunol. 5, 114–123. 25. Bonser, R. W., Thompson, N. T., Randall, R. W., and Garland, L. G. (1989) Biochem. J. 264, 617–620. 26. Whatmore, J., Cronin, P., and Cockcroft, S. (1994) FEBS Letts. 352, 113–117.
27. Agwu, D. E., McPhail, L. C., Sozzani, S., Bass, D. A., and McCall, C. E. (1991) J. Clin. Invest . 88, 531–539. 28. Rossi, F., Greskowiak, M., Della Blanca, V., Calzetti, F., and Gandini, G. (1990) Biochem. Biophys. Res. Commun. 168, 320– 327. 29. Bourgoin, S., Borgeat, P., and Poubelle, P. E. (1991) Agents Actions 34, 32–34. 30. Bourgoin, S., Poubelle, P. E., Liao, N. W., Umezawa, K., Borgeat, P., and Naccache, P. H. (1992) Cell. Signalling 4, 487–500. 31. Uings, I. J., Thompson, N. T., Randall, R. W., Spacey, G. D., Bonser, R. W., Hudson, A. T., and Garland, L. G. (1992) Biochem. J . 281, 597–600. 32. Berkow, R. L., and Dodson, R. W. (1990) Blood 75, 2445–2452. 33. Lloyds, D., and Hallett, M. B. (1994) Biochem. Pharmacol. 48, 15–21. 34. Dusi, S., Domini, M., Dellabianca, V., Gandini, G., and Rossi, F. (1994) Biochem. Biophys. Res. Commun. 201, 30–37. 35. Hamada, F., Aoki, M., Akiyama, T., and Toyoshima, K. (1993) Proc. Natn. Acad. Sci. USA 90, 6305–6309.
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