Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0806536 on October 24, 2006

Published online before print October 24, 2006
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(Journal of Leukocyte Biology. 2007;81:567-577.)
© 2007 by Society for Leukocyte Biology

Differential involvement of NF-{kappa}B and MAP kinase pathways in the generation of inflammatory cytokines by human neutrophils

Alexandre Cloutier1, Thornin Ear1, Emilie Blais-Charron, Claire M. Dubois and Patrick P. McDonald2

Pulmonary Division, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada

2 Correspondence: Pulmonary Division, Faculty of Medicine, Université de Sherbrooke, 3001, 12e avenue Nord, pièce 4849, Sherbrooke, Québec, Canada J1H 5N4. E-mail: patrick.mcdonald{at}usherbrooke.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of human neutrophils to express a variety of genes encoding inflammatory mediators is well documented, and mounting evidence suggests that neutrophil-derived cytokines and chemokines contribute to the recruitment of discrete leukocyte populations at inflammatory sites. Despite this, our understanding of the signaling intermediates governing the generation of inflammatory cytokines by neutrophils remains fragmentary. Here, we report that inhibitors of the p38 MAPK and MEK pathways substantially diminish the release of (and in the case of p38 inhibitors, the gene expression of) several inflammatory cytokines in neutrophils stimulated with LPS or TNF. In addition, various NF-{kappa}B inhibitors were found to profoundly impede the inducible gene expression and release of inflammatory cytokines in these cells. The MAPK inhibitors did not affect NF-{kappa}B activation; instead, the transcriptional effects of the p38 MAPK inhibitor appear to involve transcriptional factor IID. Conversely, the NF-{kappa}B inhibitors failed to affect the activation of MAPKs. Finally, the MAPK inhibitors were found to prevent the activation a key component of the translational machinery, S6 ribosomal protein, in keeping with their post-transcriptional impact on cytokine generation. To our knowledge, this constitutes the first demonstration that in neutrophils, the inducible expression of proinflammatory cytokines by physiological stimuli largely reflects the ability of the latter to activate NF-{kappa}B and selected MAPK pathways. Our data also raise the possibility that NF-{kappa}B or MAPK inhibitors could be useful in the treatment of inflammatory disorders in which neutrophils predominate.

Key Words: transcription factors • protein kinases • chemokines • neutrophils • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In recent years, the ability of neutrophils to synthesize several immunoregulatory proteins has forced a reassessment of their contribution to host defense processes. In addition to their traditional role as professional phagocytes, it has become evident that neutrophils can modulate ongoing inflammatory or immune processes through the production of a wide variety of cytokines and chemokines (reviewed in ref. [1 ]). However, despite a growing body of knowledge about which immunoregulatory proteins are produced by neutrophils, as well as which stimuli induce their synthesis, the mechanisms underlying this response remain, at best, partially understood. With few exceptions, the inducible production of cytokines and chemokines by neutrophils is preceded by an accumulation of the corresponding mRNA transcripts (reviewed in ref. [1 ]). In the specific cases of IL-1ß, CXCL8 (IL-8), and CCL3 (MIP-1{alpha}), we and others [2 3 4 5 ] have further established that this inducible mRNA accumulation principally reflects an increased transcription of the corresponding genes, as opposed to changes in mRNA stability. Together, these observations emphasize the central role of transcriptional events in the induction of cytokine and chemokine production in human neutrophils. In this regard, it is noteworthy that the genes encoding TNF-{alpha}, IL-1ß, CXCL8, CCL3, and CCL4 all contain {kappa}B (or {kappa}B-like) motifs in their promoter region and that the binding of NF-{kappa}B dimers to these motifs suffices to confer transcriptional inducibility (reviewed in ref. [6 ]). In addition, classical stimuli of NF-{kappa}B activation (such as LPS or TNF-{alpha}) have been shown to increase the transcription of the genes encoding the above cytokines and chemokines in neutrophils [3 4 5 ]. Finally, we demonstrated in a series of articles that many classes of agonists, known for their ability to induce the production of inflammatory cytokines and chemokines in neutrophils, can also activate NF-{kappa}B in these cells [7 8 9 10 ]. Collectively, these considerations strongly suggest a participation of transcription factor NF-{kappa}B in the transcriptional activation of cytokine and chemokine genes in neutrophils. To date, however, this still awaits a formal demonstration.

In addition to their ability to activate transcription factors, inducers of inflammatory cytokine production in neutrophils mobilize other signaling cascades, which could impact on this response. It is known, for instance, that stimuli such as LPS and TNF can activate the p38 MAPK and MEK/ERK pathways in neutrophils [11 12 13 ]. In this regard, we and others [14 15 16 17 ] have shown that in LPS-stimulated neutrophils, the p38 MAPK pathway is involved in CXCL8 and TNF-{alpha} release. Similarly, CXCL8 secretion is diminished by MEK inhibitors in LPS-activated neutrophils [15 , 17 ]. By contrast, we and others [12 , 13 , 18 19 20 21 22 23 24 25 26 27 ] have shown that the JNK pathway is not mobilized upon neutrophil activation under most experimental conditions with the exception of adherent neutophils, in which JNK can be activated moderately [27 , 28 ]. Accordingly, we have shown that a JNK inhibitor does not diminish inflammatory cytokine production by neutrophils [27 ]. Thus, although a picture is slowly emerging about the potential involvement of signaling pathways in the onset of inflammatory cytokine generation by neutrophils, much remains to be learned. In particular, it is still unclear whether MAPK involvement is limited to cytokine release or if it also affects gene expression. Similarly, the eventual impact of these signaling pathways on transcriptional events (such as NF-{kappa}B activation) remains to be established.

In the present study, we investigated whether inhibition of selected MAPK pathways and of the NF-{kappa}B pathway alters the inducible expression of several inflammatory cytokines and chemokines in primary human neutrophils. We now report that cytokine release is profoundly affected by inhibitors of the p38 MAPK and MEK/ERK cascades and that this largely mirrors an effect on inducible gene expression in the case of p38 MAPK inhibition. We also report that various NF-{kappa}B blockers strongly interfere with inflammatory cytokine gene expression and release in neutrophils and that this effect did not appear to involve any cross-talk with the MAPK pathways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and reagents
Antibodies raised against p38 MAPK, ERK1/2, and S6 ribosomal protein, as well as against their phospho forms (#9211, 9212, 9101, 9102, 2211) were from Cell Signaling Technology (Beverly, MA); antibodies against I{kappa}B{alpha} (sc-371) and transcriptional factor IID (TFIID; sc-421) were from Santa Cruz Biotechnology (CA). Ficoll-Paque, T4 polynucleotide kinase, and poly (dI-dC) were from Amersham-Pharmacia (Baie d’Urfé, Québec, Canada); [{gamma}-32P]-adenosine 5'-triphosphate (ATP) and [{alpha}-32P]-uridine 5'-triphosphate were from New England Nuclear (Boston, MA). Endotoxin-free (<2 pg/ml) RPMI 1640 and FCS were from Sigma-Aldrich (St. Louis, MO) and Hyclone (Logan, UT), respectively. Recombinant human cytokines were from R&D Systems (Minneapolis, MN), and LPS (from Escherichia coli 0111:B4) was from List Biological Laboratories (Campbell, CA). Acetylated BSA, diisopropyl fluorophosphate (DFP), fMLP, PMSF, and pyrrolidine dithiocarbamate (PDTC) were from Sigma-Aldrich. PGA1 and PGE2 as well as 15-deoxy-PGJ2 were purchased from Cayman (Ann Arbor, MI). The protease inhibitors aprotinin, 4-(2-aminomethyl)benzenesulfonyl fluoride (AEBSF), leupeptin, and pepstatin A were from Roche (Laval, Québec, Canada). Inhibitors of NF-{kappa}B (MG-132, MG-262, BAY 117082) and of MAPKs (SB203580, PD98059) were from EMD Biosciences (San Diego, CA). All other reagents were of the highest available grade, and all buffers and solutions were prepared using pyrogen-free clinical grade water.

Cell isolation and culture
Neutrophils were isolated from the peripheral blood of healthy donors, following a protocol that was duly approved by an institutional ethics committee. The entire procedure was carried out at room temperature and under endotoxin-free conditions using a modification of the method of Boyum [29 ]. Briefly, blood was collected by venepuncture and spun at 200 g for 10 min; plasma was removed carefully and replaced with sterile PBS. Following Dextran sedimentation, cells were centrifuged over Ficoll-Paque cushions; the resulting PBMC ring was carefully collected, and the erythrocytes remaining in the neutrophil pellet were removed by hypotonic lysis with water (20 s). Purified neutrophils were resuspended in RPMI 1640, supplemented with 10% FCS at a final concentration of 5 x 106 cells/ml (unless otherwise stated). As determined by Wright staining and nonspecific esterase cytochemistry, the final neutrophil suspensions consistently contained fewer than 0.5% monocytes or lymphocytes; unless otherwise stated, neutrophil viability exceeded 98% after up to 4 h in culture, as determined by trypan blue exclusion.

EMSA
Cells were incubated at 37°C in the presence or absence of stimuli. Incubations were stopped by adding equivalent volumes of ice-cold PBS, supplemented with DFP (2 mM, final concentration) and phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4, 10 mM Na4P2O7) prior to centrifugation at 300 g for 5 min at 4°C. Cells were resuspended in ice-cold relaxation buffer (10 mM PIPES, pH 7.30, 10 mM NaCl, 3.5 mM MgCl2, 0.5 mM EGTA, 0.5 mM EDTA, 1 mM DTT), supplemented with an antiprotease cocktail (1 mM DFP, 1 mM PMSF, 1 mM AEBSF, and 10 µg/ml each aprotinin, leupeptin, and pepstatin A, final concentrations) and the aforementioned phosphatase inhibitors. Nuclear extracts were then prepared using a nitrogen bomb procedure, which we described previously [7 , 9 ]. The nuclear extracts were analyzed subsequently in EMSA for NF-{kappa}B binding as described earlier [7 ]. Similar binding conditions were used when samples were analyzed using the TATA-containing TFIID probe, 5'-gcagagcaTATAAAAtgaggtagga-3' (Santa Cruz Biotechnology).

Denaturing electrophoreses and immunoblots
For whole-cell samples, incubations were stopped as described above; a small aliquot was taken from each sample for subsequent protein content determination, and neutrophils were then pelleted (200 g, 10 min). Boiling sample buffer was added directly to the cell pellets, which were vortexed briefly and placed in boiling water for a further 3 min. Samples thus prepared were sonicated to disrupt chromatin and stored at –20°C prior to analysis. When cytoplasmic and nuclear fractions were prepared, incubations were stopped as described above, and neutrophils were disrupted by nitrogen cavitation as described previously [7 , 9 ]. This procedure was also shown to yield nuclear and cytoplasmic fractions, which are exempt from cross-contamination [7 , 30 ]. After taking a small aliquot from each sample (for subsequent protein content determination in Bradford assays), concentrated sample buffer (prewarmed at 95°C) was added directly to cytoplasmic or nuclear fractions [to yield a final concentration of 1x sample buffer, i.e., 25 mM TrisBase, pH 6.80, 2% sodium dodecylsulfate (w/v), 5% 2-ME (v/v), 10% glycerol (v/v)] prior to a 3-min incubation at 95°C. All samples were electrophoresed on denaturing gels prepared according to the method of Laemmli [31 ]; equal loading was ascertained by adjusting sample volumes based on their respective protein content. Following SDS-PAGE, proteins were transferred onto nitrocellulose membranes, which were stained with Ponceau Red, destained, and then processed for immunoblot analysis as described previously [32 ].

Kinase assays
Neutrophils were incubated in the presence or absence of SB203580 for 30 min at 37°C and stimulated with LPS, TNF, or diluent control for 10 min. Cells were disrupted by NP-40 lysis as described before [7 , 9 ], and cytoplasmic fractions were immunoprecipitated with anti-p38 MAPK antibodies in Lysis buffer supplemented with NaCl (to final concentrations of 1% and 150 mM, respectively). Immunoprecipitates were washed three times in Lysis buffer (with NP-40 and NaCl) and finally resuspended in 20 µl kinase buffer (20 mM HEPES, pH 7.2, 10 mM MgCl2, 5 mM DTT, 20 nM ATP, 0.05% NP-40) along with 80 ng activating transcription factor 2 (ATF-2; 1–96) fragment (Santa Cruz Biotechnology) per reaction and 0.5 µl [32P]ATP (10 mCi/ml). The resulting mixtures were incubated for 15 min at 37°C; reactions were stopped by the addition of boiling Laemmli sample buffer 2x, and samples were analyzed on SDS-PAGE prior to gel-drying and autoradiography of the dried gels.

Isolation of RNA and RNase protection assays
Neutrophils were incubated in the presence or absence of stimuli or inhibitors for the desired times, as indicated. Total RNA was extracted following a slightly modified Chomczynski and Sacchi procedure [33 ] and analyzed by RNase protection assay as described previously [27 ] using multiprobe templates from BD PharMingen (Mississauga, Ontario, Canada).

ELISA analysis of secreted proteins
Neutrophils (3x106 cells/600 µl) were cultured in 12-well culture plates at 37°C under a 5% CO2 atmosphere in the presence or absence of stimuli and/or inhibitors for the indicated times. Culture supernatants as well as the corresponding cell pellets were carefully collected, snap-frozen in liquid nitrogen, and stored at –70°C. Cytokine concentrations were determined in in-house sandwich ELISA assays using commercially available capture and detection antibody pairs (R&D Systems, BD PharMingen). Detection limits using these assays varied between 3 and 10 pg/ml.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of the NF-{kappa}B pathway in activated neutrophils
We first assessed the ability of several NF-{kappa}B inhibitors to prevent NF-{kappa}B activation in neutrophils, as a prerequisite step to using the inhibitors on functional responses. Neutrophils were therefore stimulated with LPS or TNF, following pretreatment with the proteasome inhibitors MG-132 and MG-262, which prevent I{kappa}B{alpha} degradation [34 , 35 ], with BAY 117082, which prevents I{kappa}B{alpha} phosphorylation [36 ], with the cyclopentenone prostanoids PGA1 and 15-deoxy-PGJ2, which inhibit I{kappa}B kinase (IKK)ß specifically [37 ], or with PDTC, an antioxidant that was reported to inhibit NF-{kappa}B activation in several cell lines [38 ]. Neutrophils were subsequently stimulated with LPS or TNF prior to EMSA analysis. All of the above inhibitors were found to effectively prevent NF-{kappa}B activation with varying potencies (Fig. 1 and data not shown). Inhibitor concentrations yielding an optimal NF-{kappa}B blockade were thus selected for the subsequent experiments (10 µM for MG-132; 1 µM for MG-262; 5 µM for BAY 117082; 30 µM for 15-deoxy-PGJ2). In the particular case of PDTC, a pronounced inhibition required that the cells be exposed to at least 300 µM (Fig. 1B , right panel), a rather high concentration, which was also found to diminish neutrophil viability in culture (by ~15% after 2 h). It is noteworthy that when nuclear extracts from LPS-treated neutrophils were coincubated in the EMSA-binding mix with individual NF-{kappa}B blockers (at the same concentrations that were deemed optimal for NF-{kappa}B inhibition), no inhibition of NF-{kappa}B DNA binding was observed (data not shown). This therefore rules out the possibility that the NF-{kappa}B blockers might somehow interfere directly with the EMSA analyses.


Figure 1
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  		Figure 1. Effect of NF-{kappa}B inhibitors on inducible NF-{kappa}B DNA binding in neutrophils. Cells were stimulated for 15 min with 100 ng/ml LPS, 100 U/ml TNF-{alpha}, or diluent control, following a pretreatment (30 min) with the indicated concentrations of MG-132 or BAY 117082 (A) or a longer pretreatment (60 min) with the indicated concentrations of PGA1, 15-deoxy-PGJ2, or PDTC (B). Nuclear extracts were then prepared and analyzed in EMSA using a NF-{kappa}B oligonucleotide probe. The experiments shown in this figure are representative of at least three.

 
Effect of NF-{kappa}B blockade on inducible cytokine expression in neutrophils
We next investigated the consequence of inhibiting the NF-{kappa}B pathway on the ability of neutrophils to express inflammatory cytokines. Cells were pretreated with the various NF-{kappa}B blockers prior to stimulation with LPS or TNF, and ribonuclease protection assay (RPA) analysis of cytokine mRNA accumulation was performed. As shown in Figure 2 , the NF-{kappa}B inhibitors strongly repressed the inducible expression of all inflammatory cytokines investigated, although this inhibition was consistently less marked in the case of CXCL8 gene expression. By contrast, the constitutive TGF-ß1 mRNA levels, which are detected in resting and activated neutrophils [39 , 40 ], were unaffected by any of the NF-{kappa}B-interfering agents used (data not shown), in keeping with the fact that basal TGF-ß expression is largely independent of NF-{kappa}B activity. The above results were confirmed further by re-analyzing selected experiments by real-time RT-PCR (data not shown). In agreement with our RNA data, the NF-{kappa}B blockers were found to profoundly affect the release into culture supernatants of the corresponding cytokines in LPS- and TNF-stimulated neutrophils (Fig. 3 ). These results establish that several inflammatory cytokines produced by neutrophils are under the control of NF-{kappa}B.


Figure 2
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  		Figure 2. Effect of NF-{kappa}B inhibitors on inflammatory cytokine gene expression in neutrophils. Cells were pretreated with various NF-{kappa}B inhibitors as described in the legend to Figure 1 prior to stimulation for 60 min with 100 ng/ml LPS, 100 U/ml TNF-{alpha}, or diluent control. Total RNA was then extracted and analyzed in RPA. Mean ± SEM from at least three independent experiments.

 

Figure 3
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  		Figure 3. Effect of NF-{kappa}B inhibitors on inflammatory cytokine production in neutrophils. Cells were pretreated with various NF-{kappa}B inhibitors as described in the legend to Figure 1 prior to stimulation for 5 h with 100 ng/ml LPS, 100 U/ml TNF-{alpha}, or diluent control. Culture supernatants were then collected and analyzed in ELISA. Mean ± SEM from at least five independent experiments.

 
Blockade of the ERK and p38 MAPK pathways in activated neutrophils
In contrast with the NF-{kappa}B pathway, we recently established that the JNK/AP-1 cascade does not participate in the induction of inflammatory cytokines and chemokines in neutrophils under most circumstances [27 ]. To investigate the involvement of the other main MAPK cascades in this response, namely the p38 MAPK and MEK/ERK pathways, we treated neutrophils with highly selective inhibitors prior to stimulation and determination of functional responses. Dose-response experiments were initially conducted to determine the appropriate inhibitor concentrations to be used. To assess the effect of the MEK-1 inhibitor, PD98059, cells were preteated with the drug and stimulated with the strong MEK and ERK activator, fMLP [41 ]. As shown in Figure 4A , low micromolar concentrations of PD98059 potently inhibited MEK activation (as determined by ERK1/2 phosphorylation), and 10 µM PD98059 consistently yielded a nearly complete inhibition, as reported previously [41 , 42 ]. We next assessed the effect of the p38 MAPK inhibitor SB203580 in neutrophils treated with the strong p38 MAPK activator, LPS [12 ]. As shown in Figure 4B , pretreatment with increasing concentrations of SB203580 progressively interfered with p38 MAPK activation by LPS or TNF-{alpha} (Fig. 4B) , an almost complete inhibition being consistently achieved using 3 µM SB203580. The above experiments also confirmed the selectivity of the chosen MAPK pathway inhibitors, insofar as SB203580 (at 3 µM) failed to affect ERK activation, and conversely, PD98059 (up to 20 µM) did not prevent p38 MAPK activation (Fig. 4B , lane 3, and data not shown). Thus, PD98059 was used at a concentration of 10–20 µM in the remainder of our study to effectively inhibit the MEK/ERK pathway, and SB203580 was used at 3 µM to block the p38 MAPK pathway.


Figure 4
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  		Figure 4. Effect of MAPK inhibitors on p38 MAPK and MEK activation in neutrophils, which (A) were pretreated for 30 min with increasing concentrations of PD98059 prior to stimulation with 30 nM fMLP for 10 min. The cells were then disrupted and processed for immunoblot analysis using antiphospho-ERK (P-ERK) antibodies. The membranes were subsequently stripped and reprobed with ERK1/2 antibody. (B) Neutrophils were pretreated for 30 min with increasing concentrations of SB203580 or with 20 µM PD98059 (lane 3) prior to stimulation with 100 ng/ml LPS for 10 min. The cells were then disrupted, and p38 MAPK was immunoprecipitated from the lysates. The resulting immunoprecipitates were split in equal parts, which were analyzed immediately in kinase assays using recombinant ATF-2 as substrate, or processed for SDS-PAGE and subsequent immunoblot analysis of their p38 MAPK content. The experiments shown in this figure are representative of three.

 
Effect of MEK and p38 MAPK inhibition on inducible cytokine expression in neutrophils
We next examined the effect of the MAPK inhibitors on the expression of inflammatory cytokines in neutrophils. As shown in Figure 5 , cell pretreatment with SB203580 profoundly inhibited the generation of TNF-{alpha}, CXCL8, CCL3, and CCL4 in response to LPS or TNF. The production of these various chemokines was also inhibited by PD98059, albeit to a lesser extent than in neutrophils exposed to the p38 MAPK inhibitor (Fig. 5) . As the generation of inflammatory cytokines by neutrophils is largely dependent on the induction of the corresponding genes (as reviewed in ref. [1 ]), we also investigated the effect of inhibiting the p38 MAPK and MEK/ERK pathways on cytokine gene expression. As shown in Figure 6 , the p38 MAPK inhibitor significantly hindered the inducible expression of CXCL8, CCL3, and CCL4 in neutrophils, albeit to a lesser extent than cytokine secretion. In contrast, cell pretreament with the ERK pathway inhibitor had little effect on cytokine gene expression (Fig. 6) . Again, re-analysis of selected experiments by real-time RT-PCR yielded similar results, thereby confirming the outcome of our RNase protection analyses (data not shown).


Figure 5
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  		Figure 5. Effect of MAPK inhibitors on inflammatory cytokine production in neutrophils. Cells were pretreated (30 min, 37°C) with 3 µM SB203580 or 20 µM PD98059 prior to stimulation for 5 h with 100 ng/ml LPS, 100 U/ml TNF-{alpha}, or diluent control. Culture supernatants were then collected and analyzed in ELISA. Mean ± SEM from at least five independent experiments.

 

Figure 6
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  		Figure 6. Effect of MAPK inhibitors on inflammatory cytokine gene expression in neutrophils. Cells were pretreated (30 min, 37°C) with 3 µM SB203580 or 20 µM PD98059 prior to stimulation for 60 min with 100 ng/ml LPS, 100 U/ml TNF-{alpha}, or diluent control. Total RNA was then extracted and analyzed in RPA. Mean ± SEM from at least three independent experiments, except for TNF-{alpha} mRNA (two experiments).

 
Lack of cross-talk between the MAP kinase and NF-{kappa}B pathways in neutrophils
As the impediment of cytokine production by the p38 MAPK inhibitor appears to be partially transcriptional and as NF-{kappa}B activation was found to represent a key transcriptional event for cytokine production, we investigated whether there might be a cross-talk between these pathways. Neutrophils were therefore preincubated with increasing concentrations of the MAPK inhibitors prior to stimulation with LPS or TNF, and NF-{kappa}B activation was then assessed in EMSA. Figure 7A , left panel, shows that up to 20 µM PD98059 had no effect on NF-{kappa}B activation under all conditions tested. Similarly, neutrophil pretreatment with up to 3 µM SB203580 consistently failed to affect NF-{kappa}B activation. However, higher concentrations (10 µM or more) interfered strongly with the induction of NF-{kappa}B DNA binding by LPS or TNF (Fig. 7A , right panel), as previously reported [14 ]. Given that SB203580 blocks over 90% of p38 MAPK activity at a concentration of 3 µM in neutrophils (Fig. 4) , these results indicate that the inhibition of NF-{kappa}B activation observed at higher SB203580 concentrations probably represents a nonspecific effect. We conclude that the inhibitory effect of MAPK blockers on cytokine gene expression in neutrophils cannot be attributed to an inhibition of NF-{kappa}B by these compounds. Conversely, NF-{kappa}B inhibition had no effect on the activation of p38 MAPK or ERK2, as determined by their inducible phosphorylation in response to LPS or TNF (Fig. 7B) . Thus, the MAPK pathways appear to affect cytokine production by neutrophils independently of NF-{kappa}B, and vice versa.


Figure 7
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  		Figure 7. Lack of cross-talk between the MAPK and NF-{kappa}B pathways in neutrophils. (A) Cells were pretreated (30 min, 37°C) with 20 µM PD98059 (PD; left panel) or with increasing concentrations of SB203580 (right panel) prior to stimulation for 10 min with 100 ng/ml LPS or diluent control. Nuclear extracts were then prepared and analyzed in EMSA using a NF-{kappa}B oligonucleotide probe. (B) Neutrophils were pretreated in the presence or absence of MG-132 (10 µM, 30 min), MG-262 (1 µM, 30 min), 15-deoxy-PGJ2 (30 µM, 60 min), PGA1 (50 µM, 60 min), or BAY117082 (10 µM, 30 min) prior to stimulation with 100 ng/ml LPS for 10 min. Cellular levels of phosphorylated or total p38 MAPK and ERK were then determined by immunoblot analysis. The experiments shown in this figure are representative of at least three.

 
Potential downstream targets of the MAPK pathways controlling inflammatory cytokine production
Our p38 MAPK inhibitor data suggested the involvement of transcriptional events other than NF-{kappa}B activation, and in this regard, it has been reported that p38 MAPK blockade can prevent the binding of TFIID to the TATA box on gene promoters in mouse macrophages [43 ]. We therefore examined whether the MAPK inhibitors could interfere with TFIID binding in neutrophils stimulated with LPS or TNF. As shown in Figure 8A , neutrophil activation leads to enhanced binding of a complex to a TATA-containing sequence in EMSA (lanes 1 and 2), and prior treatment of the cells with the p38 MAPK inhibitor (but not with the MEK inhibitor) prevented the induction of this DNA-binding activity in response to LPS or TNF. The specificity of this inducible complex was ascertained in competition experiments, which showed that low amounts of unlabeled competitor oligonucleotide in the binding mix efficiently displaced the inducible complex (Fig. 8B , lane 2), whereas much greater competitor concentrations were needed to interfere with the binding of faster-migrating complexes (Fig. 8B , lane 1). Supershift experiments confirmed the identity of the specific, inducible complex, as its binding was displaced by anti-TFIID antibodies (Fig. 8B , lane 4), and isotype-matched control IgG had no effect (Fig. 8B , lane 5). Thus, the blockade of inducible TFIID binding to its cognate sequence using inhibitors of p38 MAPK (but not of MEK) agrees well with the effect of these inhibitors on cytokine gene expression in neutrophils.


Figure 8
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  		Figure 8. MAPK targets, which potentially regulate cytokine generation in neutrophils. (A) Cells were pretreated (30 min, 37°C) with 20 µM PD98059, 3 µM SB203580 (SB), or diluent control prior to stimulation with LPS for 10 min. Nuclear extracts were then processed for EMSA analysis using a TFIID oligonucleotide probe. (B) Nuclear extracts from LPS-stimulated cells were incubated for 20 min in binding buffer with a 50- or tenfold molar excess of unlabeled probe (cold, lanes 1 and 2), with an anti-TFIID antibody (lane 4) or with an isotype-matched control IgG (lane 5), or without cold competitors or antibodies (LPS, lane 3) prior to the addition of a labeled TFIID probe and subsequent EMSA analysis. (C) Neutrophils were pretreated (30 min, 37°C) with 20 µM PD98059, 3 µM SB203580, or diluent control (–) prior to stimulation with LPS for 15 or 30 min. Cellular levels of phosphorylated S6 ribosomal protein (ser 235/236) or p38 MAPK (as a loading control) were then determined by immunoblot analysis. As a positive control for phosphorylated S6 ribosomal protein, we also included cellular extracts from LPS-activated RAW 264.7 mouse macrophages (RAW). The experiments shown in this figure are representative of at least three.

 
Our MAPK inhibitor experiments (Figs. 5 and 6) also showed that the p38 MAPK and MEK pathways affect neutrophil cytokine generation by post-transcriptional mechanisms. This is consistent with several studies, which have established that both MAPK pathways can influence the activity of various components of the translational machinery [44 45 46 ]. We therefore searched for translational intermediates that could act as targets for the MAPK pathways under study. For this purpose, neutrophils were pretreated with individual MAPK inhibitors and stimulated with LPS or TNF, and the phosphorylation of known translational intermediates was examined by immunoblot. As shown in Figure 8C , neutrophil activation leads to the time-dependent detection of phosphorylated S6 ribosomal protein, a phenomenon that was largely prevented by inhibitors of the p38 MAPK or MEK pathways. In view of the key role played by S6 ribosomal protein in the initiation of mRNA translation [47 ], these results suggest a potential basis for the post-transcriptional effect of the MAPK inhibitors toward neutrophil cytokine production.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We showed previously that most of the stimuli that can elicit cytokine and chemokine production in neutrophils also have the ability to promote NF-{kappa}B activation in these cells [7 8 9 10 ]. We now report that NF-{kappa}B activation is a central event in the basal and inducible expression of various inflammatory cytokines in human neutrophils, and that cytokine generation in these cells is also under the influence of distinct MAPK pathways. A schematic overview of these findings is presented in Figure 9 .


Figure 9
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  		Figure 9. Schematic representation of the signaling pathways participating in inflammatory cytokine generation by neutrophils and some of their molecular targets. We characterized the IKK pathway of neutrophils in a previous report [30 ]. Similarly, we determined previously that JNK is only moderately activated in neutrophils and that its inhibition does not attenuate cytokine production [27 ].

 
Although the inducible expression of inflammatory cytokines could be presumed to involve NF-{kappa}B in neutrophils, such an involvement had, thus far, remained in need of a formal demonstration. Moreover, studies performed in other cell types have revealed that the induction of some inflammatory cytokine genes, although involving NF-{kappa}B, often requires the concurrent participation of other transcription factors, as in the case of CXCL8 [48 , 49 ]. As a result, the extent of the NF-{kappa}B contribution to cytokine generation in neutrophils was also in need of clarification. To tackle this issue, we used a number of structurally unrelated NF-{kappa}B inhibitors, which all proved effective in preventing inducible NF-{kappa}B binding in neutrophils. This is consistent with our recent demonstration that these compounds also prevent I{kappa}B{alpha} degradation in neutrophils [30 ]. We showed that pretreatment of neutrophils with NF-{kappa}B blockers profoundly inhibits the induction of early-response genes encoding TNF-{alpha}, IL-1ß, CXCL8, CCL3, and CCL4 in neutrophils and hinders the release of the corresponding proteins. Thus, NF-{kappa}B appears to be a key player in the induction of inflammatory cytokines and chemokines in neutrophils. This conclusion is supported further by the selectivity of the NF-{kappa}B blockers, which had no effect on genes that are not under the direct control of NF-{kappa}B, such as the one encoding TGF-ß1. Likewise, the expression of the GAPDH and 18S genes was unaffected by the NF-{kappa}B blockers when quantitated against one another. Finally, our data are consistent with the two existing studies in which NF-{kappa}B was targeted in neutrophils. In one of them, the cross-linking of integrins on the neutrophil surface was shown to induce several responses, including NF-{kappa}B activation and the secretion of IL-1ß and TNF-{alpha}, which were hindered in cells pretreated with the NF-{kappa}B inhibitors MG-132 or SN50 [50 ]. In the second study, a fusion peptide made of a sequence within the viral Tat protein and of a sequence that binds to IKK{gamma} (thereby preventing its intereaction with IKKß) inhibited NF-{kappa}B activation and I{kappa}B{alpha} gene expression in response to LPS stimulation and accelerated constitutive neutrophil apoptosis [51 ]. Thus, the NF-{kappa}B transcription factor emerges as a key element for physiologically relevant, functional responses (i.e., inflammatory cytokine generation) and cellular processes (i.e., spontaneous apoptosis) of human neutrophils.

In addition to its reliance on NF-{kappa}B, inflammatory cytokine production was shown to be regulated by distinct MAPK pathways. Indeed, we showed that inhibition of the p38 MAPK and MEK/ERK pathways substantially diminished the release of all cytokines investigated in neutrophils stimulated with LPS or TNF. Our results therefore confirm and significantly extend earlier studies, which had shown that in LPS-treated neutrophils, p38 MAPK and MEK inhibition diminishes the release of CXCL8 [15 16 17 ]. They also confirm our earlier report that p38 MAPK inhibitors attenuate the release of TNF-{alpha} in LPS-stimulated neutrophils [14 ]. Worthy of note is that in all of these earlier studies, CXCL8 or TNF-{alpha} release was inhibited to a greater extent than reported here using the p38 MAPK inhibitor SB203580. This probably reflects the fact that higher doses of the drug were used (i.e., 10 µM or more), which we showed herein to inhibit NF-{kappa}B activation in addition to p38 MAPK activity and which were also reported to interfere with ERK activation [52 ]. By comparison, the inhibitory effect of the MAPK blockers observed herein cannot be attributed to a nonspecific inhibition of NF-{kappa}B by these compounds. This conclusion is further supported by our recently published finding, that concentrations of SB203580 and PD98059 identical to the ones used in the present study, also fail to affect the phosphorylation of individual IKK subunits and of RelA and I{kappa}B{alpha} phosphorylation and degradation [30 ]. Further investigation of the effect of MAPK blockade on inflammatory cytokine generation in neutrophils revealed that the resulting inhibition of cytokine release was mirrored by a similar (albeit less pronounced) decrease in the inducible expression of the cytokines under study in the case of p38 MAPK inhibition, whereas cytokine expression was largely unaffected by the MEK inhibitor. This indicates that transcriptional events participate in the regulation of inflammatory cytokine generation by the p38 MAPK pathway. Although our data exclude a role for NF-{kappa}B in this transcriptional regulation, a likely candidate is TFIID, as its binding to its cognate TATA-containing sequence was blocked by the p38 MAPK inhibitor, whereas it was unaffected by the MEK inhibitor. This is reminiscent of similar observations made in mouse macrophages, which had shown that p38 MAPK inhibition fails to block NF-{kappa}B activation but reduces the transactivation of {kappa}B-responsive genes by interfering with the phosphorylation of TFIID, thereby preventing it from binding to the TATA box on gene promoters [43 ]. This being said, transcriptional events only partially account for the effect of p38 inhibition of cytokine generation in neutrophils, and our data imply a role for additional regulatory steps at the level of translation and/or secretion. In this regard, we showed herein that p38 MAPK and MEK inhibitors interfered with the inducible phosphorylation of the S6 ribosomal protein, which plays an important role in the initiation of mRNA translation [47 ]. These results agree well with the post-transcriptional effect of the MAPK inhibitors and suggest that the S6 ribosomal protein may mediate, at least in part, the effect of the MAPK inhibitors toward neutrophil cytokine production. Studies are in progress to identify the various translational targets of the MAPKs and to determine their individual impact on cytokine production in human granulocytes.

Collectively, our findings provide novel insights into the mechanisms governing an important functional response of primary human neutrophils. Indeed, they constitute the first demonstration that in these cells, the generation of inflammatory cytokines by physiological stimuli largely reflects the ability of the latter to activate the NF-{kappa}B pathway and distinct MAPK cascades. Moreover, we showed that there is no cross-talk between the NF-{kappa}B and MAPK pathways, so that their respective input is complementary, and some targets of the MAPKs are emerging. In a broader context, our data raise the possibility that NF-{kappa}B or MAPK inhibitors could be useful in the treatment of chronic inflammatory disorders in which neutrophils predominate.


   ACKNOWLEDGEMENTS
 
This work was supported by grants to P. P. McD. from the Canadian Institutes of Health Research (CIHR) and the Canadian Foundation for Innovation. P. P. McD. is a scholar of the Fonds de la Recherche en Santé du Québec (FRSQ). A. C. is the recipient of a Studentship from the CIHR.


   FOOTNOTES
 
1 These authors contributed equally to this work. Back

Received August 28, 2006; revised October 5, 2006; accepted October 6, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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