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Activation of Arachidonate Release and Cytosolic Phospholipase A2 via Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase in Macrophages Stimulated by Bacteria or Zymosan
Cell. Signal. Vol. 11, No. 12, pp. 863–869, 1999 Copyright 1999 Elsevier Science Inc.
ISSN 0898-6568/99 $ – see front matter PII S0898-6568(99)00058-3
Activation of Arachidonate Release and Cytosolic Phospholipase A2 via Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase in Macrophages Stimulated by Bacteria or Zymosan Go¨sta Hiller and Roger Sundler* Department of Cell and Molecular Biology, Section for Molecular Pathogenesis, Lund University, P.O. Box 94, S-221 00 Lund, Sweden
ABSTRACT. The mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase (ERK) and p38, can both contribute to the activation of cytosolic phospholipase A2 (cPLA2). We have investigated the hypothesis that ERK and p38 together or independent of one another play roles in the regulation of cPLA2 in macrophages responding to the oral bacterium Prevotella intermedia or zymosan. Stimulation with bacteria or zymosan beads caused arachidonate release and enhanced in vitro cPLA2 activity of cell lysate by 1.5- and 1.7-fold, respectively, as well as activation of ERK and p38. The specific inhibitor of MAP kinase kinase, PD 98059, and the inhibitor of p38, SB 203580, both partially inhibited cPLA2 activation and arachidonate release induced by bacteria and zymosan. Together, the two inhibitors had additive effects and completely blocked cPLA2 activation and arachidonate release. The present results demonstrate that ERK and p38 both have important roles in the regulation of cPLA2 and together account for its activation in P. intermedia and zymosan-stimulated mouse macrophages. cell signal 11;12:863–869, 1999. 1999 Elsevier Science Inc. All rights reserved. KEY WORDS. Periodontitis, Prevotella intermedia, Protein kinases, 85-kDa phospholipase A2, Mouse macrophages, Arachidonic acid
INTRODUCTION Macrophages have many important roles in the defence against bacteria and other infectious agents. Not only do they phagocytose and kill microorganisms but they also respond by generating inflammatory mediators, such as cytokines and eicosanoids. The intracellular signalling system that is activated upon contact with certain bacteria, has been demonstrated to cause phosphorylation and activation of cytosolic phospholipase A2 (cPLA2), that hydrolyses arachidonic acid from membrane phospholipids [1]. Evidence has also been provided that activation of mitogen-activated protein kinases (MAP kinases) are involved in the generation of proinflammatory agents like cytokines [2]. One type of MAP kinase, namely 42-kDa MAP kinase or extracellular signal-regulated kinase-2 (ERK-2) was early on proposed to cause phosphorylation and activation of cPLA2 [3, 4]. Hazan et al. [5] showed that zymosan-induced cPLA2 activation could be inhibited by a MAP kinase kinase (MEK) in-
hibitor, PD 98059, in neutrophils. Later studies on hematopoetic cells and certain cell lines, using the p38 MAP kinase inhibitor SB 203580, have instead suggested more prominent roles for p38 [6, 7] in the activation of cPLA2 induced by tumour necrosis factor-a [8], collagen [9], and thrombin [10]. The purpose of the present study was therefore to investigate bacteria- and zymosan-induced arachidonic acid mobilisation and cPLA2 activation in mouse macrophages and to correlate this to the activation of the kinases ERK or p38. In these studies the Gram-negative bacterium, Prevotella intermedia, involved in periodontitis in humans [11–13], was used and compared with the more well known stimulus zymosan as reference. We found that the bacteria as well as zymosan caused activation of the MAP kinases ERK and p38 and induced cPLA2 activation and arachidonate release. The use of the selective protein kinase inhibitors PD 98059 and SB 203580 led to the conclusion that both ERK and p38 participate in the activation of cPLA2 in mouse macrophages.
*Author to whom correspondence should be addressed. Tel: 146-46-2228584; fax: 146-46-157-756; E-mail: [email protected] Abbreviations: ATF2—activating transcription factor 2; ERK—extracellular signal-regulated kinase; LPS—lipopolysaccharide; MAP kinase— mitogen-activated protein kinase; MBP—myelin basic protein; MEK— MAP kinase kinase; P. intermedia—Prevotella intermedia; cPLA2—cytosolic phospholipase A2. Received 28 April 1999; Accepted 26 July 1999.
MATERIALS AND METHODS Materials Female outbred NMRI mice were from Bom-Mice, Copenhagen, Denmark. Activating transcription factor 2 (ATF2)
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and antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). [5,6,8,9,11,12,14,15-3H] Arachidonic acid (218 Ci/mmol) and 1-stearoyl-2-[1-14C]arachidonoyl phosphatidylcholine (55 mCi/mmol) were from Amersham International (Little Chalfont, UK). PD 98059 was obtained from New England Biolabs, Inc. (Beverly, MA, USA) and SB 203580 was a gift from SmithKline Beecham Pharmaceuticals (USA). PD 98059 and SB 203580 were dissolved in DMSO and added to the cells resulting in a final DMSO concentration of 0.2%, which had no effects on the cellular responses measured. Myelin basic protein (MBP) (M-1891) and zymosan were obtained from Sigma (St. Louis, MO, USA). Protein A Sepharose CL-4B was from Pharmacia Biotech (Uppsala, Sweden) and PMA was from ICN Biomedical Inc. (Aurora, OH, USA). Prof. Stig Edwardsson (Dept. of Oral Microbiology, School of Dentistry, Malmo¨, Sweden) kindly provided bacteria, including P. intermedia diluted in PBS. Isolation of Peritoneal Macrophages Peritoneal macrophages were isolated from female outbred NMRI mice by washing with Medium 199 (Earle’s salts supplemented with 10 mM Hepes) together with 1% heatinactivated foetal bovine serum. The macrophages were isolated from the peritoneal cells by adherence to either 3.8- or 10-cm2 tissue culture dishes (Nunclon, Nunc, Roskilde, Denmark). The cells were incubated at 378C in an atmosphere of 5% CO2 in air and non-adherent cells were removed after 2 hours by washing with Dulbecco’s PBS, (pH 7.4). Medium 199 supplemented with 10% foetal bovine serum was then added to the cell cultures, which were incubated for 18–20 h.
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Laemmli sample buffer [14]. The samples were then boiled for 5 minutes and subjected to SDS-PAGE using 10% acrylamide. Gels were equilibrated in transfer buffer, consisting of 25 mM Tris, 192 mM Glycine and 20% MeOH, (5–10 min) and the proteins transferred to PVDF membranes. The membranes were then blocked in 3% gelatine for 2 h followed by incubation for 12–15 h with polyclonal ERK-2 antibodies in 1% gelatine. Antibodies bound were detected with secondary horseradish peroxidase-conjugated antibodies and the enhanced chemiluminescence system.
PLA2-Assay Macrophages (2.5 3 106 cells/well) were isolated and cultured on 10-cm2 tissue dishes as described above. Following stimulation the cells were washed twice with ice-cold PBS and scraped off the dishes in 400 ml of a buffer consisting of 80 mM KCl, 10 mM Hepes pH 7.4, 1 mM EDTA, 25 mM b-glycerophosphate, 10 mM NaF, 0.1 mM ammonium vanadate. Cells were sonicated and centrifuged for 1 h at 105 3 g to obtain a membrane pellet and a cytosol fraction. Glycerol, (10% by weight), was then added to the cytosol fraction. Aliquots (50 ml), equivalent to 4 mg protein or 0.4 3 106 cells, of the cytosol fractions were incubated for 15 min at 378C in the presence of the substrate 1-stearoyl-2 [1-14C]arachidonoyl phosphatidylcholine (100 pmol/incubation), 1.4 mM free Ca21, 5.7 mM DTE and 0.4 mg/ml BSA and the assay was performed as previously described [15]. Protein content in the cytosol fraction was determined according to the method of Bradford [16] using BSA as standard.
Immunoprecipitation and MAP Kinase Assay Arachidonate Release In experiments where the mobilisation of arachidonic acid from cellular phospholipids was determined, cells (2 3 106 cells/well) were radiolabelled with [3H]arachidonic acid (0.5 mCi) for 18–20 h. After radiolabelling, the cells were washed with PBS and exposed in 1 ml serum-free Medium 199 to bacteria (approximately 1 3 108 bacteria/ml) or zymosan (0.2 mg/ml). At the end of the experiment the medium was collected and the cells were scraped off the dish in 1.0 ml of 0.1% Triton X-100 in H2O. The medium was then centrifuged and the arachidonate released from cellular phospholipids was quantified and expressed as a percentage of the total recovered radioactivity. In experiments where PD 98059 or SB 203580 was added, cells were pretreated with 1 mM indomethacin. Immunoblotting After the culture period the cells (2.5 3 106 cells/well) were stimulated with bacteria or zymosan. At the end of the experiment the medium was withdrawn and the cells were washed twice with cold PBS and scraped off the wells in
Cells were isolated and cultured in 10-cm2 tissue culture dishes (3 3 106 cells/well) as described above. After stimulation with bacteria or zymosan the medium was removed and the cells were washed with cold PBS and scraped off the dishes in 500 ml of a lysis buffer consisting of 50 mM Hepes pH 7.4, 1 mM EDTA, 0.25 mM okadaic acid, 50 mM b-glycerophosphate, 0.1 mM ammonium vanadate, 1 mg/ml pepstatin A, 1 mg/ml aprotinin, 1mM PMSF and 1% Triton X-100. After centrifugation at 9000 3 g for 10 min the supernatant was incubated for 3 h at 48C with a mixture of polyclonal ERK-2 antibody (4 mg IgG) and protein A Sepharose (40 ml, of a 50% slurry), that had already been preincubated for 15 h at 48C. The immunoprecipitates were washed four times with lysis buffer and were then assayed for kinase activity in a buffer consisting of 40 mM MgCl2, 10 mCi [32P]ATP, 40 mg MBP and 4 mM ATP and 2.6 mM EDTA for 30 min at 378C. The reactions were stopped by addition of Laemmli sample buffer and the samples were subjected to SDS/PAGE. The gel was dried and submitted to autoradiography. Immunoprecipitation of p38 and assay of this kinase was performed in the same way as described above for ERK-2 except that a polyclonal p38 antibody (4
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mg IgG) was used for precipitation and the MBP substrate was replaced by 1.5 mg ATF2 in the kinase assay. RESULTS P. intermedia and Zymosan Induce Arachidonate Release in Mouse Macrophages Initially a wide range of different Gram-negative bacterial species related to periodontal diseases, such as Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Eikenella corrodens and Wolinella recta, were tested for their ability to cause release of [3H]arachidonate in mouse macrophages, but only some of them, in particular P. intermedia, caused pronounced release and was therefore selected for more thorough investigation. Exposure of macrophages to P. intermedia led to 8% release of cellular [3H]arachidonate above control level at 80 min and levelled off after that time (Fig. 1A). The release of radiolabel was very low during the first 10–20 min of stimulation. Bacteria that had been killed by sonication retained activity, although somewhat reduced. The concentration used (108 bact/ml) was optimal (not shown). Zymosan stimulation, which led to a more pronounced response than that induced by bacteria, amounted to a release of 33% [3H]arachidonate above control at 80 min and was initiated within 10 min. Activation of cPLA2 To test whether cPLA2 activation preceded arachidonate release we investigated the time-dependent changes in cPLA2 activity in response to bacteria and zymosan. We measured the activity of cPLA2 from bacteria- or zymosanstimulated macrophages in an in vitro assay. The results showed that P. intermedia and zymosan caused cPLA2 activation in the cytosol of macrophages after 20 and 10 min, respectively (Fig. 1B). This time course correlates in time with the arachidonate release induced by the two stimuli. Higher concentrations of P. intermedia did not cause further increase in the cPLA2 activity. Activation of ERK and p38 and Effects of PD 98059 and SB 203580 on MAP Kinase Activation To determine whether any of the MAP kinases ERK or p38 preceded cPLA2 activation induced by bacteria or zymosan, we investigated their activity as a function of time. ERK activity was assessed with an anti-phospho-ERK-2 antibody, which could cross-react with ERK-1, and we measured p38 activity by immunoprecipitation followed by in vitro kinase assay using ATF2 as substrate. P. intermedia induced a weak ERK activation after 2–10 min stimulation and the activity peaked at 20 min (Fig. 2A). Zymosan induced a slight increase in ERK activity after 2 min with progressive increase until 20 min. Both the bacteria- and the zymosan-induced ERK activity decreased to basal levels after 120 min (not shown). After 2 min stimulation of macrophages with bacteria or zymosan there was also a detectable increase in p38 activity (Fig. 2B). This activity peaked at 20 and 10 min for
FIGURE 1. Time course of [3H]arachidonate release and cPLA2
activation induced by bacteria and zymosan. (A) Macrophages were prelabeled with [3H]arachidonic acid (0.5 mCi) for 20 h. Prevotella intermedia (1 3 108 bact/ml) or zymosan (0.2 mg/ml) were then added for the time indicated. The release of radiolabel into the media is expressed as percentage of total cellular [3H], corrected for the release in control cultures (3.7 6 0.3). The data are from three (zymosan) and eight (bacteria) separate experiments and values shown are means with positive and negative S.E.M, respectively. (B) The cells were prepared and stimulated as above and fractionated into a membrane pellet and a cytosol fraction. The cytosol fraction was assayed for cPLA2 activity as described in Materials and Methods. Phospholipase activity in non-treated cells (4.0 6 0.8 pmol/mg/15 min) was set to 100% and the results represent mean and negative S.E.M of three separate experiments.
bacteria and zymosan, respectively, and then slowly decreased. However, the activity was still strong at 60 min with both stimuli. To assess the efficiency of the MEK-1/2 inhibitor PD 98059 on bacteria and zymosan-induced ERK activation, we preincubated cells with PD 98059 followed by stimulation with bacteria and zymosan. ERK was immunoprecipi-
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FIGURE 2. Temporal activation of ERK and p38 by P. interme-
dia or zymosan. (A) Bacteria (1 3 108 bacteria/ml) or zymosan (0.2 mg/ml) were added to cultures of macrophages for the indicated time. The medium was removed and Laemmli sample buffer was then added and equal aliquots of whole cell extracts were run on SDS-PAGE followed by Western blotting, using an antibody directed against phospho-ERK-2. The results are representative of three separate experiments. (B) Cells were prepared as above up to the removal of the culture medium. p38 was immunoprecipitated from the cytosol fraction and equal aliquots of protein from each sample was used for an in vitro kinase assay, using ATF2 as substrate. Laemmli sample buffer was added and the samples submitted to SDS-PAGE. The gel was dried and submitted to autoradiography.
tated from the cell lysate and the activity determined in an in vitro kinase assay with MBP as substrate. MBP phosphorylation induced by bacteria and zymosan was reduced, compared to basal levels, with 95 6 5.0% (n 5 3) and 85 6 6.5% (n 5 3), respectively, (quantitated by Phosphoimage analysis) when cells were preincubated with 10 or 50 mM PD 98059 (Fig. 3A). The zymosan-induced MBP phosphorylation was reduced by 74%, when cells were preincubated with 10 mM PD 98059 (not shown). Activation of p38 MAP kinase was measured in the same manner as ERK activation with the exception that SB 203580 was added to the in vitro kinase assay and ATF2 was used as substrate. The bacteria- and zymosan-induced ATF2 phosphorylation was completely abolished when the immunoprecipitates were treated with 10 mM SB 203580 (Fig. 3B). This is in agreement with other studies that have shown that 10 mM SB 203580 is sufficient to inhibit fully the activity of p38 both in vitro [17] and in vivo [18]. We also performed experiments where we tested whether PD 98059 had effects on p38 activation and whether SB 203580 had effects on ERK activation. Surprisingly we found that PD 98059, at a concentration of 50 mM, decreased the zymosan-induced p38 activation by 53% but was not affected by 5 and 10 mM PD 98059 (Fig. 4). Bacteria-induced stimulation of p38 was also unaffected by 5 and 10 mM PD 98059 but was even more inhibited (by 87%) in the presence of 50 mM PD 98059 (Fig 4). We measured the lactate dehydrogenase activity in the extracellular media as a measure on cellular integrity and could not detect an increased release of lactate dehydroge-
FIGURE 3. Effects of PD 98059 and SB 203580 on ERK and
p38 activation. (A) Macrophages were pretreated with or without PD 98059 (10 or 50 mM) for 10 min and then stimulated by P. intermedia (1 3 108 bacteria/ml) or zymosan (0.2 mg/ml) for 40 min. The cell lysate samples were immunoprecipitated with an ERK-2 antibody and kinase activity determined with MBP as substrate as described in Materials and Methods. (B) Cells were treated as above with the exceptions that p38 was immunoprecipitated with a p38 antibody and 10 mM SB 203580 was added to the in vitro kinase assay. The p38 activity was determined using ATF2 as substrate. 0.2% DMSO was added as vehicle. The results are representative of three separate experiments.
nase from cells incubated with PD 98059 and stimulus. This indicates that PD 98059 is either an unspecific inhibitor at higher concentrations (50 mM) or that there is one or more signalling pathways connecting MEK and/or ERK with p38. No effects on ERK activation could be seen with 10 mM SB 203580 (not shown). Effect of PD 98059 and SB 203580 on Arachidonate Release Induced by P. intermedia and Zymosan We were interested to see whether the P. intermedia and zymosan-induced release of [3H] arachidonate could be affected by the MEK and p38 MAP kinase inhibitors PD 98059 and SB 203580. As SB 203580 and PD 98059 have also been reported to inhibit cyclooxygenase-1 and -2 [19], we included the cyclooxygenase inhibitor indomethacin (1 mM) in the culture medium in all arachidonate release experiments where PD 98059 and SB 203580 were used. The release of [3H] arachidonate from cells stimulated with bacteria or zymosan for 60 min was decreased by 90 and 65%, respectively, in the presence of 50 mM PD98059 (Fig. 5A). However, the ERK activity induced by bacteria could be totally inhibited at a concentration of 10 mM PD 98059 which only reduced the bacteria-induced [3H] arachidonate release by 60%. The further decrease seen with 50 mM PD 98059 may be due, not only to ERK inhibition, but also to a decrease in p38 activity as noted above. SB 203580 (10 mM) decreased the bacterium and zymosan-induced release of arachidonate by 50 and 40%, respectively (Fig. 5B).
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FIGURE 4. Effects of PD 98059 on p38 activation. (A) Macro-
phages were treated with or without the indicated concentration of PD 98059 for 10 min and then stimulated with zymosan (0.2 mg/ml) or P. intermedia (1 3 108 bact/ml) for 20 min. The cell lysates were immunoprecipitated with a p38 antibody and kinase activity determined with ATF2 as substrate. (B) The rate of phosphorylation was quantitated by Phosphoimage analysis and presented as p38 kinase activity relative to the kinase activity in the absence of PD 98059 and stimulus (100%). The results are presented as mean and positive S.E.M. from three separate experiments.
Higher concentrations of SB 203580 (20–30 mM) did not lead to any further decrease in [3H]arachidonate release (not shown). A combination of the two MAP kinase inhibitors decreased further the bacteria- and zymosan-induced release of arachidonate (Table 1) compared to the effects seen with only PD 98059 or SB 203580 (Fig. 5). We also conducted experiments with 0.1% BSA in the media as arachidonic acid acceptor. The bacteria- and zymosan-induced [3H]arachidonate release was totally inhibited by a combination of PD 98059 and SB 203580 even in the presence of BSA in the media (not shown). This further emphasises that the effects of PD 98059 and SB 203580 do not reflect inhibition of cyclooxygenase. Effects of PD 98059 and SB 203580 on the cPLA2 Activation Induced by Bacteria and Zymosan Incubation of macrophages with 10 mM PD 98059 or 10 mM SB 203580 decreased the bacteria-induced cPLA2 activation by <50% (Table 2). Treatment of the macrophages with a combination of the two inhibitors completely
FIGURE 5. Effects of PD 98059 and SB 203580 on [3H]arachi-
donate release. Macrophages were labelled with [3H]arachidonic acid (0.5 mCi) for 20 h and were then preincubated with 1 mM indomethacin for 5 min and then indicated concentration of PD 98059 for 10 min (A) or SB 203580 for 15 min (B). The cells were stimulated with P. intermedia or zymosan for 60 min. The release of [3H]arachidonate is expressed as percent of the maximal response (set to 100%), (8.6 6 1.8, for bacteria and 20.8 6 2.1, for zymosan; mean 6 S.E.M. n 53) corrected for the release in control cultures (3.6 6 0.6).
blocked the activation of cPLA2. In the presence of 50 mM PD 98059 bacteria-induced cPLA2 activation was almost completely inhibited but again this strong inhibition is likely to be in part due to inhibitory effects on p38. The zymosan-induced cPLA2 activation was inhibited by <40% with 10 mM SB 203580 and by 50% with 50 mM PD 98059. Treatment of the cells with both inhibitors totally abolished the zymosan-induced cPLA2 activation.
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G. Hiller and R. Sundler TABLE 1. Combined effects of PD 98059 and SB 203580 on (3H) arachidonate releasea
[3H]arachidonate release (% of total [3H])
Treatment
No addition
SB 203580 (10 mM) 1 PD98059 (50 mM)
Zymosan P. intermedia
19.6 6 2.5 7.3 6 0.4
2.6 6 0.9 –
SB 203580 (10 mM) 1 PD 98059 (10 mM) – 0.7 6 0.2
Macrophages were labelled with [3H]arachidonic acid (0.5 mCi) for 20 h. The cells were preincubated with 1 mM indomethacin for 5 min and then incubated with both PD 98059 (10 or 50 mM) and SB 203580 (10 mM) for 10 min followed by stimulation with either bacteria or zymosan for 60 min. The release of radiolabel is expressed as percentage of total cellular [3H], corrected for the release in control cultures (3.8 6 0.4%, n 5 4). The results are as mean 6 S.E.M. from 4 separate experiments.
a
DISCUSSION Activation of cPLA2 by phosphorylation is considered, together with increases in intracellular Ca21, to be rate-limiting steps in the release of arachidonic acid. In this work we show that an oral bacterium (P. intermedia) like yeastderived zymosan beads cause activation of cPLA2 and arachidonate release in macrophages. While zymosan is already known to cause an increase in cytosolic calcium, activation of cPLA2 and arachidonic acid release [20, 21], it is yet not clear whether the bacteria used here cause changes in intracellular calcium. Gram-negative bacteria contain LPS in their outer membrane and may therefore stimulate cells in a way similar to pure LPS. Bacteria are, however, particulate and expose numerous potential ligands and may interact with several surface components on macrophages thereby activating additional signalling pathways. Recent data do show that P. intermedia induces increased formation of inositolphosphates (unpublished observation) which may lead to an increase in intracellular Ca21. The lag period seen in cPLA2 activation and arachidonate release induced by bacteria and zymosan is most likely due to the time it takes for bacteria and zymosan particles to sediment and contact the monolayer of macrophages. Although a weak ERK and p38 activation can be seen after
2–10 min, a much larger activation occurs later. This time course differs from several reports on MAP kinase activation by soluble agonists. If physical factors constitute the rate-limiting step in the macrophage response to the particulate stimuli studied here, it is not surprising that it is difficult to delineate the order of signalling events (i.e., whether activation of one or both of the MAP kinases really precedes cPLA2 activation). The inhibitor PD 98059 has turned out to be a potent and selective inhibitor of MEK [22] and Alessi et al. [23] have reported that 50 mM PD 98059 does not affect p38 activity in vitro. However, the bacteria- and zymosan-induced p38 activation in intact macrophages was reduced by treatment with 50 mM PD 98059. The activation of p38 induced by bacteria was more strongly reduced by 50 mM PD 98059 than that induced by zymosan. This is consistent with the more pronounced effect of 50 mM PD 98059 on bacteriainduced arachidonate release (Fig. 5A). The inhibitor of MEK has been shown to inhibit cPLA2 activation induced by opsonized zymosan in neutrophils [5]. However, thrombin- and collagen-induced phosphorylation of cPLA2 was not altered in the presence of PD 98059 in human platelets and SB 203580 was shown to inhibit the collagen-induced cPLA2 activation in human platelets [9, 24,
TABLE 2. Inhibition of cPLA2 activation by PD 98059 and SB 203580a
cPLA2 activity (% of control) Zymosan No addition SB 203580 (10 mM) PD 98059 (10 mM) PD 98059 (50 mM) PD 98059 (10 mM) 1 SB 203580 (10 mM) PD 98059 (50 mM) 6 SB 203580 (10 mM)
6 6 6 6 – 102 6
167 141 142 131
6 6b 15b 6b 12*
P. intermedia 151 124 122 111 98
6 6 6 6 6 –
4 6c 6c 6 6**
Macrophages were pretreated for 10 min with 10 or 50 mM PD 98059, 15 min with 10 mM SB 203580 or a combination of the two inhibitors before the cells were stimulated with P. intermedia or zymosan for 60 min. The cells were sonicated and fractionated into a membrane pellet and a cytosol fraction. The cytosol fractions were assayed for cPLA2 activity as described in Materials and Methods. The data are expressed as percent of the control, 4.4 6 0.7 pmol/mg protein/15 min which was set to 100%. The results are means 6 S.E.M. of three separate experiments. Statistical significance by Student’s t-test: * P , 0.01 compared to cells stimulated with zymosan and only treated with SB 203580 or PD 98059 (b). ** P , 0.01 compared to cells stimulated with P. internedia and only treated with SB 203580 or PD 98059 10 mM (c).
a
MAP Kinases and Arachidonate Mobilisation in Macrophages
25]. Also, in human neutrophils treated with tumour necrosis factor a, the activation of cPLA2 appeared to be regulated by p38 rather than ERK [8]. It has been proposed that ERK-2 may phosphorylate Ser505 and cause activation of cPLA2 [3]. However, p38 MAP kinase shares the substrate sequence preference with ERK and may therefore also be able to phosphorylate Ser-505 of cPLA2. Moreover, it has been shown that there are additional serine residues phosphorylated on macrophage cPLA2, not the least near the C-terminus [26]. Thrombin stimulates phosphorylation of Ser-505 and one or more C-terminal serine, possibly Ser-727 on cPLA2 and phosphorylation on both sites can be decreased by SB 203580 on cPLA2 in human platelets [27]. However, cross-linking of the low-affinity IgG receptor Fcg-RII leads to cPLA2 phosphorylation that is decreased by both PD 98059 and SB 203580 [9]. This latter finding is consistent with the possibility that both ERK and p38 contribute to the activation of cPLA2. In the present work we have been using both inhibitors to determine the engagement of MAP kinases in the regulation of cytosolic PLA2 in macrophages. Inhibition of ERK with PD 98059 did reduce the cPLA2 activation induced by both bacteria and zymosan. This was accompanied by a similar reduction in arachidonate release. SB 203580, on the other hand, also inhibited bacteria- and zymosan-induced cPLA2 activation and mobilisation of arachidonate by about 50%. When both inhibitors were used together cPLA2 activation and arachidonate release were totally inhibited. These results indicate that both ERK and p38 are involved in the regulation of cPLA2 activity in macrophages and that they together fully account for the activation caused by the bacterium P. intermedia and zymosan particles. We thank Pia Lundqvist for excellent technical assistance. The authors also thank Prof. Stig Edwardsson and Birgitta Lindberg for providing us with bacteria. This work was supported by grants from the Swedish ¨ sterlund Medical Research Council (project no. 5410), the Alfred O Foundation, Albert Pa˚hlsson Foundation, the Greta and Johan Kock Foundations, King Gustaf V 80-year Foundation, the Thelma Zoe´ga Foundation, the Crafoord Foundation, the Royal Physiographic Society and the Faculties of Odontology and Medicine, Lund University.
References 1. Svensson U., Houweling M., Holst E. and Sundler R. (1993) Eur. J. Biochem. 213, 81–86.
869 2. Lee J. C., Laydon J. T., McDonnell P. C., Gallagher T. F., Kumar S., Green D., McNulty D., Blumenthal M. J., Heys J. R., Landvatter S. W., Strickler J. E., McLaughlin M. M., Siemens I. R., Fisher S. M., Livi G. P., White J. R., Adams J. L. and Young P. R. (1994) Nature (Lond.) 372, 739–745. 3. Lin L.-L., Wartmann M., Lin A. Y., Knopf J. L., Seth A. and Davis R. J. (1993) Cell 72, 269–278. 4. Nemenoff R. A., Winitz S., Qian N.-X., Van Putten V., Johnson G. L. and Heasley L. E. (1993) J. Biol. Chem. 268, 1960– 1964. 5. Hazan I., Dana R., Granot Y. and Levy R. (1997) Biochem. J. 326, 867–876. 6. Han J., Lee J.-D., Bibbs L. and Ulevitch R. J. (1994) Science 265, 808–811. 7. Rouse J., Cohen P., Trigon S., Morange M., Alonso-Llamazares A., Zamanillo D., Hunt T. and Nebreda A. R. (1994) Cell 78, 1027–1037. 8. Waterman W. H., Molski T. F. P., Huang C.-K., Adams J. L. and Sha’afi R. I. (1996) Biochem. J. 319, 17–20. 9. Bo¨rsch-Haubold A. G., Kramer R. M. and Watson S. P. (1997) Eur. J. Biochem. 245, 751–759. 10. Herna´ndez M., Bayo´n Y., Sa´nchez Crespo M. and Nieto M. L. (1997) Biochem. J. 328, 263–269. 11. Loesche W. J., Syed S. A., Laughon B. E. and Stoll J. (1982) J. Periodontol. 53, 223–230. 12. Slots J. and Listergarten M. A. (1988) J. Clin. Periodontol. 15, 85–93. 13. Lopez N. J., Mellado J. C. and Leighton G. X. (1996) J. Clin. Periodontol. 23, 101–105. 14. Laemmli U. K. (1970) Nature (Lond.) 227, 680–685. 15. Wijkander J. and Sundler R. (1989) FEBS Lett. 244, 51–56. 16. Bradford M. M. (1976) Anal. Biochem. 72, 248–254. 17. Enslen H., Raingeaud J. and Davis R. J. (1998) J. Biol. Chem. 273, 1741–1748. 18. Cuenda A., Rouse J., Doza Y. N., Meier R., Cohen P., Gallagher T. F., Young P. R. and Lee J. C. (1995) FEBS Lett. 229–233. 19. Bo¨rsch-Haubold A. G., Pasquet S. and Watson S. P. (1998) J. Biol. Chem. 273, 28766–28772. 20. Qiu Z.-H., Gijo´n M. A., de Carvalho M. S., Spencer D. M. and Leslie C. C. (1998) J. Biol. Chem. 273, 8203–8211. 21. Emilsson A. and Sundler R. (1986) Biochem. Biophys. Acta 876, 533–542. 22. Dudley D. T., Pang L., Decker S. J., Bridges A. J. and Saltiel A. R. (1995) Proc. Natl. Acad. Sci. USA 92, 7686–7689. 23. Alessi D. R., Cuenda A., Cohen P., Dudley D. T. and Saltiel A. R. (1995) J. Biol. Chem. 270, 27489–27494. 24. Kramer R. M., Roberts E. F., Um S. L., Bo¨rsch-Haubold A. G., Watson S. P., Fisher M. J. and Jakubowski J. A. (1996) J. Biol. Chem. 271, 27723–27729. 25. Bo¨rsch-Haubold A. G., Kramer R. M. and Watson S. P. (1996) Biochem. J. 318, 207–212. 26. Wijkander J., Gewert K., Svensson U., Holst E. and Sundler R. (1997) Biochem. J. 325, 405–410. 27. Bo¨rsch-Haubold A. G., Bartoli F., Asselin J., Dudler T., Kramer R. M., Apitz-Castro R., Watson S. P. and Gelb M. H. (1998) J. Biol. Chem. 273, 4449–4458.
ISSN 0898-6568/99 $ – see front matter PII S0898-6568(99)00058-3
Activation of Arachidonate Release and Cytosolic Phospholipase A2 via Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase in Macrophages Stimulated by Bacteria or Zymosan Go¨sta Hiller and Roger Sundler* Department of Cell and Molecular Biology, Section for Molecular Pathogenesis, Lund University, P.O. Box 94, S-221 00 Lund, Sweden
ABSTRACT. The mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase (ERK) and p38, can both contribute to the activation of cytosolic phospholipase A2 (cPLA2). We have investigated the hypothesis that ERK and p38 together or independent of one another play roles in the regulation of cPLA2 in macrophages responding to the oral bacterium Prevotella intermedia or zymosan. Stimulation with bacteria or zymosan beads caused arachidonate release and enhanced in vitro cPLA2 activity of cell lysate by 1.5- and 1.7-fold, respectively, as well as activation of ERK and p38. The specific inhibitor of MAP kinase kinase, PD 98059, and the inhibitor of p38, SB 203580, both partially inhibited cPLA2 activation and arachidonate release induced by bacteria and zymosan. Together, the two inhibitors had additive effects and completely blocked cPLA2 activation and arachidonate release. The present results demonstrate that ERK and p38 both have important roles in the regulation of cPLA2 and together account for its activation in P. intermedia and zymosan-stimulated mouse macrophages. cell signal 11;12:863–869, 1999. 1999 Elsevier Science Inc. All rights reserved. KEY WORDS. Periodontitis, Prevotella intermedia, Protein kinases, 85-kDa phospholipase A2, Mouse macrophages, Arachidonic acid
INTRODUCTION Macrophages have many important roles in the defence against bacteria and other infectious agents. Not only do they phagocytose and kill microorganisms but they also respond by generating inflammatory mediators, such as cytokines and eicosanoids. The intracellular signalling system that is activated upon contact with certain bacteria, has been demonstrated to cause phosphorylation and activation of cytosolic phospholipase A2 (cPLA2), that hydrolyses arachidonic acid from membrane phospholipids [1]. Evidence has also been provided that activation of mitogen-activated protein kinases (MAP kinases) are involved in the generation of proinflammatory agents like cytokines [2]. One type of MAP kinase, namely 42-kDa MAP kinase or extracellular signal-regulated kinase-2 (ERK-2) was early on proposed to cause phosphorylation and activation of cPLA2 [3, 4]. Hazan et al. [5] showed that zymosan-induced cPLA2 activation could be inhibited by a MAP kinase kinase (MEK) in-
hibitor, PD 98059, in neutrophils. Later studies on hematopoetic cells and certain cell lines, using the p38 MAP kinase inhibitor SB 203580, have instead suggested more prominent roles for p38 [6, 7] in the activation of cPLA2 induced by tumour necrosis factor-a [8], collagen [9], and thrombin [10]. The purpose of the present study was therefore to investigate bacteria- and zymosan-induced arachidonic acid mobilisation and cPLA2 activation in mouse macrophages and to correlate this to the activation of the kinases ERK or p38. In these studies the Gram-negative bacterium, Prevotella intermedia, involved in periodontitis in humans [11–13], was used and compared with the more well known stimulus zymosan as reference. We found that the bacteria as well as zymosan caused activation of the MAP kinases ERK and p38 and induced cPLA2 activation and arachidonate release. The use of the selective protein kinase inhibitors PD 98059 and SB 203580 led to the conclusion that both ERK and p38 participate in the activation of cPLA2 in mouse macrophages.
*Author to whom correspondence should be addressed. Tel: 146-46-2228584; fax: 146-46-157-756; E-mail: [email protected] Abbreviations: ATF2—activating transcription factor 2; ERK—extracellular signal-regulated kinase; LPS—lipopolysaccharide; MAP kinase— mitogen-activated protein kinase; MBP—myelin basic protein; MEK— MAP kinase kinase; P. intermedia—Prevotella intermedia; cPLA2—cytosolic phospholipase A2. Received 28 April 1999; Accepted 26 July 1999.
MATERIALS AND METHODS Materials Female outbred NMRI mice were from Bom-Mice, Copenhagen, Denmark. Activating transcription factor 2 (ATF2)
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and antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). [5,6,8,9,11,12,14,15-3H] Arachidonic acid (218 Ci/mmol) and 1-stearoyl-2-[1-14C]arachidonoyl phosphatidylcholine (55 mCi/mmol) were from Amersham International (Little Chalfont, UK). PD 98059 was obtained from New England Biolabs, Inc. (Beverly, MA, USA) and SB 203580 was a gift from SmithKline Beecham Pharmaceuticals (USA). PD 98059 and SB 203580 were dissolved in DMSO and added to the cells resulting in a final DMSO concentration of 0.2%, which had no effects on the cellular responses measured. Myelin basic protein (MBP) (M-1891) and zymosan were obtained from Sigma (St. Louis, MO, USA). Protein A Sepharose CL-4B was from Pharmacia Biotech (Uppsala, Sweden) and PMA was from ICN Biomedical Inc. (Aurora, OH, USA). Prof. Stig Edwardsson (Dept. of Oral Microbiology, School of Dentistry, Malmo¨, Sweden) kindly provided bacteria, including P. intermedia diluted in PBS. Isolation of Peritoneal Macrophages Peritoneal macrophages were isolated from female outbred NMRI mice by washing with Medium 199 (Earle’s salts supplemented with 10 mM Hepes) together with 1% heatinactivated foetal bovine serum. The macrophages were isolated from the peritoneal cells by adherence to either 3.8- or 10-cm2 tissue culture dishes (Nunclon, Nunc, Roskilde, Denmark). The cells were incubated at 378C in an atmosphere of 5% CO2 in air and non-adherent cells were removed after 2 hours by washing with Dulbecco’s PBS, (pH 7.4). Medium 199 supplemented with 10% foetal bovine serum was then added to the cell cultures, which were incubated for 18–20 h.
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Laemmli sample buffer [14]. The samples were then boiled for 5 minutes and subjected to SDS-PAGE using 10% acrylamide. Gels were equilibrated in transfer buffer, consisting of 25 mM Tris, 192 mM Glycine and 20% MeOH, (5–10 min) and the proteins transferred to PVDF membranes. The membranes were then blocked in 3% gelatine for 2 h followed by incubation for 12–15 h with polyclonal ERK-2 antibodies in 1% gelatine. Antibodies bound were detected with secondary horseradish peroxidase-conjugated antibodies and the enhanced chemiluminescence system.
PLA2-Assay Macrophages (2.5 3 106 cells/well) were isolated and cultured on 10-cm2 tissue dishes as described above. Following stimulation the cells were washed twice with ice-cold PBS and scraped off the dishes in 400 ml of a buffer consisting of 80 mM KCl, 10 mM Hepes pH 7.4, 1 mM EDTA, 25 mM b-glycerophosphate, 10 mM NaF, 0.1 mM ammonium vanadate. Cells were sonicated and centrifuged for 1 h at 105 3 g to obtain a membrane pellet and a cytosol fraction. Glycerol, (10% by weight), was then added to the cytosol fraction. Aliquots (50 ml), equivalent to 4 mg protein or 0.4 3 106 cells, of the cytosol fractions were incubated for 15 min at 378C in the presence of the substrate 1-stearoyl-2 [1-14C]arachidonoyl phosphatidylcholine (100 pmol/incubation), 1.4 mM free Ca21, 5.7 mM DTE and 0.4 mg/ml BSA and the assay was performed as previously described [15]. Protein content in the cytosol fraction was determined according to the method of Bradford [16] using BSA as standard.
Immunoprecipitation and MAP Kinase Assay Arachidonate Release In experiments where the mobilisation of arachidonic acid from cellular phospholipids was determined, cells (2 3 106 cells/well) were radiolabelled with [3H]arachidonic acid (0.5 mCi) for 18–20 h. After radiolabelling, the cells were washed with PBS and exposed in 1 ml serum-free Medium 199 to bacteria (approximately 1 3 108 bacteria/ml) or zymosan (0.2 mg/ml). At the end of the experiment the medium was collected and the cells were scraped off the dish in 1.0 ml of 0.1% Triton X-100 in H2O. The medium was then centrifuged and the arachidonate released from cellular phospholipids was quantified and expressed as a percentage of the total recovered radioactivity. In experiments where PD 98059 or SB 203580 was added, cells were pretreated with 1 mM indomethacin. Immunoblotting After the culture period the cells (2.5 3 106 cells/well) were stimulated with bacteria or zymosan. At the end of the experiment the medium was withdrawn and the cells were washed twice with cold PBS and scraped off the wells in
Cells were isolated and cultured in 10-cm2 tissue culture dishes (3 3 106 cells/well) as described above. After stimulation with bacteria or zymosan the medium was removed and the cells were washed with cold PBS and scraped off the dishes in 500 ml of a lysis buffer consisting of 50 mM Hepes pH 7.4, 1 mM EDTA, 0.25 mM okadaic acid, 50 mM b-glycerophosphate, 0.1 mM ammonium vanadate, 1 mg/ml pepstatin A, 1 mg/ml aprotinin, 1mM PMSF and 1% Triton X-100. After centrifugation at 9000 3 g for 10 min the supernatant was incubated for 3 h at 48C with a mixture of polyclonal ERK-2 antibody (4 mg IgG) and protein A Sepharose (40 ml, of a 50% slurry), that had already been preincubated for 15 h at 48C. The immunoprecipitates were washed four times with lysis buffer and were then assayed for kinase activity in a buffer consisting of 40 mM MgCl2, 10 mCi [32P]ATP, 40 mg MBP and 4 mM ATP and 2.6 mM EDTA for 30 min at 378C. The reactions were stopped by addition of Laemmli sample buffer and the samples were subjected to SDS/PAGE. The gel was dried and submitted to autoradiography. Immunoprecipitation of p38 and assay of this kinase was performed in the same way as described above for ERK-2 except that a polyclonal p38 antibody (4
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mg IgG) was used for precipitation and the MBP substrate was replaced by 1.5 mg ATF2 in the kinase assay. RESULTS P. intermedia and Zymosan Induce Arachidonate Release in Mouse Macrophages Initially a wide range of different Gram-negative bacterial species related to periodontal diseases, such as Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Eikenella corrodens and Wolinella recta, were tested for their ability to cause release of [3H]arachidonate in mouse macrophages, but only some of them, in particular P. intermedia, caused pronounced release and was therefore selected for more thorough investigation. Exposure of macrophages to P. intermedia led to 8% release of cellular [3H]arachidonate above control level at 80 min and levelled off after that time (Fig. 1A). The release of radiolabel was very low during the first 10–20 min of stimulation. Bacteria that had been killed by sonication retained activity, although somewhat reduced. The concentration used (108 bact/ml) was optimal (not shown). Zymosan stimulation, which led to a more pronounced response than that induced by bacteria, amounted to a release of 33% [3H]arachidonate above control at 80 min and was initiated within 10 min. Activation of cPLA2 To test whether cPLA2 activation preceded arachidonate release we investigated the time-dependent changes in cPLA2 activity in response to bacteria and zymosan. We measured the activity of cPLA2 from bacteria- or zymosanstimulated macrophages in an in vitro assay. The results showed that P. intermedia and zymosan caused cPLA2 activation in the cytosol of macrophages after 20 and 10 min, respectively (Fig. 1B). This time course correlates in time with the arachidonate release induced by the two stimuli. Higher concentrations of P. intermedia did not cause further increase in the cPLA2 activity. Activation of ERK and p38 and Effects of PD 98059 and SB 203580 on MAP Kinase Activation To determine whether any of the MAP kinases ERK or p38 preceded cPLA2 activation induced by bacteria or zymosan, we investigated their activity as a function of time. ERK activity was assessed with an anti-phospho-ERK-2 antibody, which could cross-react with ERK-1, and we measured p38 activity by immunoprecipitation followed by in vitro kinase assay using ATF2 as substrate. P. intermedia induced a weak ERK activation after 2–10 min stimulation and the activity peaked at 20 min (Fig. 2A). Zymosan induced a slight increase in ERK activity after 2 min with progressive increase until 20 min. Both the bacteria- and the zymosan-induced ERK activity decreased to basal levels after 120 min (not shown). After 2 min stimulation of macrophages with bacteria or zymosan there was also a detectable increase in p38 activity (Fig. 2B). This activity peaked at 20 and 10 min for
FIGURE 1. Time course of [3H]arachidonate release and cPLA2
activation induced by bacteria and zymosan. (A) Macrophages were prelabeled with [3H]arachidonic acid (0.5 mCi) for 20 h. Prevotella intermedia (1 3 108 bact/ml) or zymosan (0.2 mg/ml) were then added for the time indicated. The release of radiolabel into the media is expressed as percentage of total cellular [3H], corrected for the release in control cultures (3.7 6 0.3). The data are from three (zymosan) and eight (bacteria) separate experiments and values shown are means with positive and negative S.E.M, respectively. (B) The cells were prepared and stimulated as above and fractionated into a membrane pellet and a cytosol fraction. The cytosol fraction was assayed for cPLA2 activity as described in Materials and Methods. Phospholipase activity in non-treated cells (4.0 6 0.8 pmol/mg/15 min) was set to 100% and the results represent mean and negative S.E.M of three separate experiments.
bacteria and zymosan, respectively, and then slowly decreased. However, the activity was still strong at 60 min with both stimuli. To assess the efficiency of the MEK-1/2 inhibitor PD 98059 on bacteria and zymosan-induced ERK activation, we preincubated cells with PD 98059 followed by stimulation with bacteria and zymosan. ERK was immunoprecipi-
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FIGURE 2. Temporal activation of ERK and p38 by P. interme-
dia or zymosan. (A) Bacteria (1 3 108 bacteria/ml) or zymosan (0.2 mg/ml) were added to cultures of macrophages for the indicated time. The medium was removed and Laemmli sample buffer was then added and equal aliquots of whole cell extracts were run on SDS-PAGE followed by Western blotting, using an antibody directed against phospho-ERK-2. The results are representative of three separate experiments. (B) Cells were prepared as above up to the removal of the culture medium. p38 was immunoprecipitated from the cytosol fraction and equal aliquots of protein from each sample was used for an in vitro kinase assay, using ATF2 as substrate. Laemmli sample buffer was added and the samples submitted to SDS-PAGE. The gel was dried and submitted to autoradiography.
tated from the cell lysate and the activity determined in an in vitro kinase assay with MBP as substrate. MBP phosphorylation induced by bacteria and zymosan was reduced, compared to basal levels, with 95 6 5.0% (n 5 3) and 85 6 6.5% (n 5 3), respectively, (quantitated by Phosphoimage analysis) when cells were preincubated with 10 or 50 mM PD 98059 (Fig. 3A). The zymosan-induced MBP phosphorylation was reduced by 74%, when cells were preincubated with 10 mM PD 98059 (not shown). Activation of p38 MAP kinase was measured in the same manner as ERK activation with the exception that SB 203580 was added to the in vitro kinase assay and ATF2 was used as substrate. The bacteria- and zymosan-induced ATF2 phosphorylation was completely abolished when the immunoprecipitates were treated with 10 mM SB 203580 (Fig. 3B). This is in agreement with other studies that have shown that 10 mM SB 203580 is sufficient to inhibit fully the activity of p38 both in vitro [17] and in vivo [18]. We also performed experiments where we tested whether PD 98059 had effects on p38 activation and whether SB 203580 had effects on ERK activation. Surprisingly we found that PD 98059, at a concentration of 50 mM, decreased the zymosan-induced p38 activation by 53% but was not affected by 5 and 10 mM PD 98059 (Fig. 4). Bacteria-induced stimulation of p38 was also unaffected by 5 and 10 mM PD 98059 but was even more inhibited (by 87%) in the presence of 50 mM PD 98059 (Fig 4). We measured the lactate dehydrogenase activity in the extracellular media as a measure on cellular integrity and could not detect an increased release of lactate dehydroge-
FIGURE 3. Effects of PD 98059 and SB 203580 on ERK and
p38 activation. (A) Macrophages were pretreated with or without PD 98059 (10 or 50 mM) for 10 min and then stimulated by P. intermedia (1 3 108 bacteria/ml) or zymosan (0.2 mg/ml) for 40 min. The cell lysate samples were immunoprecipitated with an ERK-2 antibody and kinase activity determined with MBP as substrate as described in Materials and Methods. (B) Cells were treated as above with the exceptions that p38 was immunoprecipitated with a p38 antibody and 10 mM SB 203580 was added to the in vitro kinase assay. The p38 activity was determined using ATF2 as substrate. 0.2% DMSO was added as vehicle. The results are representative of three separate experiments.
nase from cells incubated with PD 98059 and stimulus. This indicates that PD 98059 is either an unspecific inhibitor at higher concentrations (50 mM) or that there is one or more signalling pathways connecting MEK and/or ERK with p38. No effects on ERK activation could be seen with 10 mM SB 203580 (not shown). Effect of PD 98059 and SB 203580 on Arachidonate Release Induced by P. intermedia and Zymosan We were interested to see whether the P. intermedia and zymosan-induced release of [3H] arachidonate could be affected by the MEK and p38 MAP kinase inhibitors PD 98059 and SB 203580. As SB 203580 and PD 98059 have also been reported to inhibit cyclooxygenase-1 and -2 [19], we included the cyclooxygenase inhibitor indomethacin (1 mM) in the culture medium in all arachidonate release experiments where PD 98059 and SB 203580 were used. The release of [3H] arachidonate from cells stimulated with bacteria or zymosan for 60 min was decreased by 90 and 65%, respectively, in the presence of 50 mM PD98059 (Fig. 5A). However, the ERK activity induced by bacteria could be totally inhibited at a concentration of 10 mM PD 98059 which only reduced the bacteria-induced [3H] arachidonate release by 60%. The further decrease seen with 50 mM PD 98059 may be due, not only to ERK inhibition, but also to a decrease in p38 activity as noted above. SB 203580 (10 mM) decreased the bacterium and zymosan-induced release of arachidonate by 50 and 40%, respectively (Fig. 5B).
MAP Kinases and Arachidonate Mobilisation in Macrophages
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FIGURE 4. Effects of PD 98059 on p38 activation. (A) Macro-
phages were treated with or without the indicated concentration of PD 98059 for 10 min and then stimulated with zymosan (0.2 mg/ml) or P. intermedia (1 3 108 bact/ml) for 20 min. The cell lysates were immunoprecipitated with a p38 antibody and kinase activity determined with ATF2 as substrate. (B) The rate of phosphorylation was quantitated by Phosphoimage analysis and presented as p38 kinase activity relative to the kinase activity in the absence of PD 98059 and stimulus (100%). The results are presented as mean and positive S.E.M. from three separate experiments.
Higher concentrations of SB 203580 (20–30 mM) did not lead to any further decrease in [3H]arachidonate release (not shown). A combination of the two MAP kinase inhibitors decreased further the bacteria- and zymosan-induced release of arachidonate (Table 1) compared to the effects seen with only PD 98059 or SB 203580 (Fig. 5). We also conducted experiments with 0.1% BSA in the media as arachidonic acid acceptor. The bacteria- and zymosan-induced [3H]arachidonate release was totally inhibited by a combination of PD 98059 and SB 203580 even in the presence of BSA in the media (not shown). This further emphasises that the effects of PD 98059 and SB 203580 do not reflect inhibition of cyclooxygenase. Effects of PD 98059 and SB 203580 on the cPLA2 Activation Induced by Bacteria and Zymosan Incubation of macrophages with 10 mM PD 98059 or 10 mM SB 203580 decreased the bacteria-induced cPLA2 activation by <50% (Table 2). Treatment of the macrophages with a combination of the two inhibitors completely
FIGURE 5. Effects of PD 98059 and SB 203580 on [3H]arachi-
donate release. Macrophages were labelled with [3H]arachidonic acid (0.5 mCi) for 20 h and were then preincubated with 1 mM indomethacin for 5 min and then indicated concentration of PD 98059 for 10 min (A) or SB 203580 for 15 min (B). The cells were stimulated with P. intermedia or zymosan for 60 min. The release of [3H]arachidonate is expressed as percent of the maximal response (set to 100%), (8.6 6 1.8, for bacteria and 20.8 6 2.1, for zymosan; mean 6 S.E.M. n 53) corrected for the release in control cultures (3.6 6 0.6).
blocked the activation of cPLA2. In the presence of 50 mM PD 98059 bacteria-induced cPLA2 activation was almost completely inhibited but again this strong inhibition is likely to be in part due to inhibitory effects on p38. The zymosan-induced cPLA2 activation was inhibited by <40% with 10 mM SB 203580 and by 50% with 50 mM PD 98059. Treatment of the cells with both inhibitors totally abolished the zymosan-induced cPLA2 activation.
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G. Hiller and R. Sundler TABLE 1. Combined effects of PD 98059 and SB 203580 on (3H) arachidonate releasea
[3H]arachidonate release (% of total [3H])
Treatment
No addition
SB 203580 (10 mM) 1 PD98059 (50 mM)
Zymosan P. intermedia
19.6 6 2.5 7.3 6 0.4
2.6 6 0.9 –
SB 203580 (10 mM) 1 PD 98059 (10 mM) – 0.7 6 0.2
Macrophages were labelled with [3H]arachidonic acid (0.5 mCi) for 20 h. The cells were preincubated with 1 mM indomethacin for 5 min and then incubated with both PD 98059 (10 or 50 mM) and SB 203580 (10 mM) for 10 min followed by stimulation with either bacteria or zymosan for 60 min. The release of radiolabel is expressed as percentage of total cellular [3H], corrected for the release in control cultures (3.8 6 0.4%, n 5 4). The results are as mean 6 S.E.M. from 4 separate experiments.
a
DISCUSSION Activation of cPLA2 by phosphorylation is considered, together with increases in intracellular Ca21, to be rate-limiting steps in the release of arachidonic acid. In this work we show that an oral bacterium (P. intermedia) like yeastderived zymosan beads cause activation of cPLA2 and arachidonate release in macrophages. While zymosan is already known to cause an increase in cytosolic calcium, activation of cPLA2 and arachidonic acid release [20, 21], it is yet not clear whether the bacteria used here cause changes in intracellular calcium. Gram-negative bacteria contain LPS in their outer membrane and may therefore stimulate cells in a way similar to pure LPS. Bacteria are, however, particulate and expose numerous potential ligands and may interact with several surface components on macrophages thereby activating additional signalling pathways. Recent data do show that P. intermedia induces increased formation of inositolphosphates (unpublished observation) which may lead to an increase in intracellular Ca21. The lag period seen in cPLA2 activation and arachidonate release induced by bacteria and zymosan is most likely due to the time it takes for bacteria and zymosan particles to sediment and contact the monolayer of macrophages. Although a weak ERK and p38 activation can be seen after
2–10 min, a much larger activation occurs later. This time course differs from several reports on MAP kinase activation by soluble agonists. If physical factors constitute the rate-limiting step in the macrophage response to the particulate stimuli studied here, it is not surprising that it is difficult to delineate the order of signalling events (i.e., whether activation of one or both of the MAP kinases really precedes cPLA2 activation). The inhibitor PD 98059 has turned out to be a potent and selective inhibitor of MEK [22] and Alessi et al. [23] have reported that 50 mM PD 98059 does not affect p38 activity in vitro. However, the bacteria- and zymosan-induced p38 activation in intact macrophages was reduced by treatment with 50 mM PD 98059. The activation of p38 induced by bacteria was more strongly reduced by 50 mM PD 98059 than that induced by zymosan. This is consistent with the more pronounced effect of 50 mM PD 98059 on bacteriainduced arachidonate release (Fig. 5A). The inhibitor of MEK has been shown to inhibit cPLA2 activation induced by opsonized zymosan in neutrophils [5]. However, thrombin- and collagen-induced phosphorylation of cPLA2 was not altered in the presence of PD 98059 in human platelets and SB 203580 was shown to inhibit the collagen-induced cPLA2 activation in human platelets [9, 24,
TABLE 2. Inhibition of cPLA2 activation by PD 98059 and SB 203580a
cPLA2 activity (% of control) Zymosan No addition SB 203580 (10 mM) PD 98059 (10 mM) PD 98059 (50 mM) PD 98059 (10 mM) 1 SB 203580 (10 mM) PD 98059 (50 mM) 6 SB 203580 (10 mM)
6 6 6 6 – 102 6
167 141 142 131
6 6b 15b 6b 12*
P. intermedia 151 124 122 111 98
6 6 6 6 6 –
4 6c 6c 6 6**
Macrophages were pretreated for 10 min with 10 or 50 mM PD 98059, 15 min with 10 mM SB 203580 or a combination of the two inhibitors before the cells were stimulated with P. intermedia or zymosan for 60 min. The cells were sonicated and fractionated into a membrane pellet and a cytosol fraction. The cytosol fractions were assayed for cPLA2 activity as described in Materials and Methods. The data are expressed as percent of the control, 4.4 6 0.7 pmol/mg protein/15 min which was set to 100%. The results are means 6 S.E.M. of three separate experiments. Statistical significance by Student’s t-test: * P , 0.01 compared to cells stimulated with zymosan and only treated with SB 203580 or PD 98059 (b). ** P , 0.01 compared to cells stimulated with P. internedia and only treated with SB 203580 or PD 98059 10 mM (c).
a
MAP Kinases and Arachidonate Mobilisation in Macrophages
25]. Also, in human neutrophils treated with tumour necrosis factor a, the activation of cPLA2 appeared to be regulated by p38 rather than ERK [8]. It has been proposed that ERK-2 may phosphorylate Ser505 and cause activation of cPLA2 [3]. However, p38 MAP kinase shares the substrate sequence preference with ERK and may therefore also be able to phosphorylate Ser-505 of cPLA2. Moreover, it has been shown that there are additional serine residues phosphorylated on macrophage cPLA2, not the least near the C-terminus [26]. Thrombin stimulates phosphorylation of Ser-505 and one or more C-terminal serine, possibly Ser-727 on cPLA2 and phosphorylation on both sites can be decreased by SB 203580 on cPLA2 in human platelets [27]. However, cross-linking of the low-affinity IgG receptor Fcg-RII leads to cPLA2 phosphorylation that is decreased by both PD 98059 and SB 203580 [9]. This latter finding is consistent with the possibility that both ERK and p38 contribute to the activation of cPLA2. In the present work we have been using both inhibitors to determine the engagement of MAP kinases in the regulation of cytosolic PLA2 in macrophages. Inhibition of ERK with PD 98059 did reduce the cPLA2 activation induced by both bacteria and zymosan. This was accompanied by a similar reduction in arachidonate release. SB 203580, on the other hand, also inhibited bacteria- and zymosan-induced cPLA2 activation and mobilisation of arachidonate by about 50%. When both inhibitors were used together cPLA2 activation and arachidonate release were totally inhibited. These results indicate that both ERK and p38 are involved in the regulation of cPLA2 activity in macrophages and that they together fully account for the activation caused by the bacterium P. intermedia and zymosan particles. We thank Pia Lundqvist for excellent technical assistance. The authors also thank Prof. Stig Edwardsson and Birgitta Lindberg for providing us with bacteria. This work was supported by grants from the Swedish ¨ sterlund Medical Research Council (project no. 5410), the Alfred O Foundation, Albert Pa˚hlsson Foundation, the Greta and Johan Kock Foundations, King Gustaf V 80-year Foundation, the Thelma Zoe´ga Foundation, the Crafoord Foundation, the Royal Physiographic Society and the Faculties of Odontology and Medicine, Lund University.
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