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The Journal of Immunology, 2003, 171: 3751-3761.
Copyright © 2003 by The American Association of Immunologists

Inflammatory Cytokine Expression Is Independent of the c-Jun N-Terminal Kinase/AP-1 Signaling Cascade in Human Neutrophils1

Alexandre Cloutier2, Thornin Ear2, Olga Borissevitch, Pierre Larivée and Patrick P. McDonald3

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the last decade, the ability of neutrophils to generate proinflammatory cytokines has become firmly established. Because neutrophils typically infiltrate inflammatory sites in large numbers, they could significantly contribute to the cytokine environment and even represent a substantial source of cytokines in chronic inflammatory disorders in which they predominate over other cell types. To date, however, most studies have focused on identifying which mediators are produced by neutrophils, as opposed to elucidating the molecular bases underlying this process. We previously showed that most stimuli of cytokine production in neutrophils also activate NF-{kappa}B in these cells. In this report, we turned our attention to another transcription factor that plays a central role in inflammation, AP-1. Among Jun/Fos proteins, only JunD and c-Fos are abundantly expressed in neutrophils, and they are mainly cytoplasmic. Both the cellular levels and distribution of the Jun/Fos proteins remain unaffected by various neutrophil stimuli, including those that are known to increase the corresponding mRNA transcripts. Similarly, c-Jun N-terminal kinase (JNK) 1 is overwhelmingly cytoplasmic in neutrophils and does not translocate to the nucleus upon cell activation. Although JNK is not activatable under most circumstances, specific conditions do allow its phosphorylation in response to TNF. However, no experimental condition (even those leading to JNK activation) resulted in the induction of genuine AP-1 complexes in neutrophils. Accordingly, the potent JNK inhibitor, SP 600125, failed to inhibit inflammatory cytokine gene expression in neutrophils. Collectively, our findings strongly suggest that the JNK/AP-1 signaling pathway has little or no impact on the generation of inflammatory mediators in neutrophils.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neutrophils have long been considered key players in the context of host defense against injury and infection. This owes much to their ability to engulf pathogenic agents, to release a battery of microbicidal compounds (including proteolytic enzymes and reactive oxygen intermediates) and to synthesize vasoactive and chemotactic lipid mediators (such as platelet-activating factor and leukotrienes). Until about a decade ago, in fact, neutrophils were essentially considered to be little more than terminally differentiated, accomplished phagocytes. Since then, a sizable number of studies have added a new dimension to our understanding of how neutrophils contribute to host immunity. Neutrophils can indeed be induced to express a number of early response genes, the products of which lie at the core of inflammatory and immune responses. These include growth factors; cytokines such as TNF-{alpha}, IL-1{beta}, IL-12, and TGF-{beta}; and a wide array of chemokines (for chemotactic cytokines) such as IL-8, growth-related oncogene-{alpha}, macrophage-inflammatory protein (Mip)4 -1{alpha}{beta}, Mip-3{alpha}{beta}, IFN-{gamma}-inducible protein (IP-10), mitogen-activated gene (MIG), IFN-{gamma}-inducible T cell {alpha} chemoattractant (I-tAc), and others (reviewed in Ref. 1). Among these mediators, chemokines are particularly relevant to inflammatory processes, because of their ability to selectively recruit discrete cell populations into sites of injury (2, 3), thereby effectively regulating leukocyte trafficking. As a result, the ability of neutrophils to produce various inflammatory mediators has far-reaching implications. In view of the fact that neutrophils are usually the first blood cells to infiltrate inflamed tissues and that they vastly outnumber other leukocytes in a variety of chronic inflammatory disorders, their ability to generate various inflammatory cytokines is likely to have a significant impact on the initiation and evolution of inflammatory reactions. Moreover, their ability to produce a plethora of chemokines raises the possibility that neutrophils might actively contribute to the sequential recruitment of distinct leukocyte populations that is typically observed in many, if not most, inflammatory reactions.

One of the recurring findings in most of the studies having addressed cytokine generation by neutrophils is that this rapidly induced response is typically preceded by (and largely dependent on) an accumulation of the corresponding mRNA transcripts (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). In the particular case of IL-1{beta}, IL-8, and Mip-1{alpha}, inducible gene expression was shown by us and others to reflect an increased transcriptional activity, as determined in nuclear run-on assays (14, 15, 16). Although these observations illustrate the critical role of transcriptional events in the context of inducible cytokine production in neutrophils, the molecular bases underlying this response have only begun to be elucidated. In this regard, we previously reported that in neutrophils, NF-{kappa}B is activated by many of the same stimuli that elicit the production of inflammatory cytokines by these cells (17, 18, 19, 20). This is in keeping with the known requirement for NF-{kappa}B for the inducible expression of many of genes encoding the aforementioned inflammatory cytokines in other cell types.

Another important transcriptional regulator for the inducible expression of many inflammatory mediators (including cytokines, growth factors, chemokines, and adhesion molecules) is AP-1. A partial list of these mediators includes TNF, IL-1, IL-1 receptor antagonist, IL-2, ICAM, tissue factor, IL-8, and monocyte chemoattractant protein (MCP)-1 (21, 22, 23, 24, 25, 26, 27, 28). The AP-1 transcription factor typically consists of combinations of Jun and Fos family proteins, which bind as dimers to a common enhancer sequence on gene promoters. Members of the Jun family of proteins (c-Jun, JunB, JunD) have the ability to homo- and heterodimerize among themselves or to dimerize with Fos family proteins, resulting in multiple AP-1 variants (29). By contrast, Fos family proteins (c-Fos, FosB, Fra-1, Fra-2) must associate with Jun proteins to form AP-1 complexes (29). Dimerization occurs via a basic leucine zipper domain within the Jun/Fos proteins, which also makes it possible for them to associate with members of different transcription factor families, such as NF-{kappa}B/Rel proteins (30), activating transcription factor (ATF)/CREB proteins (31, 32), and a host of others (33, 34). Most cell types contain some Jun/Fos proteins, and on cell stimulation, their genes are actively transcribed, an immediate-early process that usually yields more Jun/Fos proteins that are then available to form DNA-binding dimers in the nucleus. Posttranslational modification also plays a key role with regard to AP-1 function. Phosphorylation of c-Jun by c-Jun N-terminal kinase (JNK), and of c-Fos by Fyn-related kinase, has been shown to greatly potentiate the trans activation potential of AP-1 complexes (35, 36, 37). Similarly, it has been recently reported that JNKs can phosphorylate JunD and thereby enhance its ability to activate transcription (38), even though JunD was observed to bind JNK with much lower affinity than c-Jun.

This study was undertaken to delineate the role of transcription factors (other than NF-{kappa}B) in the inducible expression of inflammatory cytokine genes in human neutrophils. The rationale behind our focus on AP-1 is multifold. First, several inflammatory mediators generated by activated neutrophils are known to contain AP-1 elements within their upstream regulatory region that are either essential or needed for full promoter activity. Second, these mediators are often induced in response to known AP-1 activators in neutrophils. Third, neutrophils have been reported to constitutively express the genes encoding c-Fos, c-Jun, JunB, and JunD (39, 40, 41). The steady state level of these transcripts can be rapidly up-regulated in response to formyl peptides, GM-CSF, G-CSF, TNF, or phorbol esters in the case of c-Fos (42, 43, 44) or in response to LPS, TNF, and phorbol esters in the case of the Jun family members (41, 45). Whether the corresponding proteins are also susceptible to modulation (or whether they are expressed at all), and whether they can form functional AP-1 complexes, remains to be demonstrated in neutrophils. We now report that at least the JunD and c-Fos proteins are constitutively expressed in neutrophils but that their cellular levels are not modulated by agonists known to affect their mRNA levels. Moreover, we show that genuine AP-1 activity cannot be induced in neutrophils and that this is paralleled by a general inability of neutrophils to activate JNK under most circumstances. Thus, a role for the JNK/AP-1 signaling pathway in cytokine production by neutrophils is most unlikely.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

Purified Abs raised against proteins of the Jun and Fos families were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), as well as from Geneka (Montreal, Quebec, Canada). An oligonucleotide containing tandemly repeated NF-{kappa}B sites identical with those of the HIV promoter (5'-gatcaGGGACTTTCCgctgGGGACTTTCC-3') was synthesized, whereas an oligonucleotide containing a consensus AP-1 sequence (5'-cgcttgaTGAGTCAgccggaa-3') was from Promega (Madison, WI). Ficoll-Paque, T4 polynucleotide kinase and poly(dI-dC) were from Pharmacia (Uppsala, Sweden); [{gamma}-32P]ATP and [{alpha}-32P]UTP were from NEN (Boston, MA). Endotoxin-free (<6 pg/ml) RPMI 1640 and FCS were from BioMédia (Drummondville, Quebec, Canada) 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, cycloheximide, diisopropyl fluorophosphate (DFP), fMLP, PMA, and PMSF were from Sigma-Aldrich (St. Louis, MO). The protease inhibitors, aprotinin, 4-(2-aminomethyl)benzenesulfonyl fluoride (AEBSF), leupeptin, and pepstatin A, were all from Boehringer-Mannheim (Mannheim, Germany). Inhibitors of JNK (SP 600125) and of the proteasome (MG-132, MG-262) were from Calbiochem (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

The human promyelocytic PLB-985 cell line was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany), and was cultured at 37°C under a humidified 5% CO2 atmosphere in RPMI supplemented with 10% heat-inactivated (56°C, 30 min) FCS, 2 mM L-glutamine, and antibiotics(100 µg/ml streptomycin, 100 U/ml penicillin). Neutrophils were isolated from the peripheral blood of healthy donors under endotoxin-free conditions by a modification of the method of Boyum (46); the entire procedure was conducted at room temperature. Briefly, blood was collected by venipuncture and spun at 200 x g for 10 min; plasma was carefully removed and replaced with sterile PBS. After 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, and neutrophil viability exceeded 98% after up to 3 h in culture, as determined by trypan blue exclusion. Isolated cells were cultured in tissue culture (TC)-treated plasticware at 37°C under a humidified 5% CO2 atmosphere; in some experiments, the cells were alternatively incubated in polypropylene tubes at 37°C, with occasional agitation. Where indicated, neutrophils were resuspended at high density (2 x 107 cells/ml) and allowed to sediment for 55 min at 37°C before culture in TC-treated plasticware; this promotes cell adherence, and neutrophils thus treated are therefore referred to as adherent neutrophils.

EMSAs

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), before centrifugation at 300 x 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 mixture (1 mM DFP, 1 mM PMSF, 1 mM AEBSF, and 10 µg/ml each of 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 (17, 20). The nuclear extracts were subsequently analyzed in EMSA for NF-{kappa}B binding as described earlier (17). When EMSA analyses were performed using an AP-1 probe, a modified binding buffer was used (20 mM HEPES (pH 7.50), 50 mM KCl, 0.5 mM MgCl2, 1 mM EDTA, 5 mM DTT, 0.1% Nonidet P-40, 6% glycerol) supplemented with 0.4 µg poly(dI-dC) and 8 µg of acetylated BSA.

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 x g, 10 min). Boiling sample buffer was directly added to the cell pellets, which were briefly vortexed and placed in boiling water for a further 3 min. Samples thus prepared were sonicated to disrupt chromatin and stored at -20°C before analysis. When cytoplasmic and nuclear fractions were prepared, incubations were stopped as described above, and neutrophils were disrupted by nitrogen cavitation as described previously (17, 20). This procedure was also shown to yield nuclear and cytoplasmic fractions that are exempt from cross-contamination (17, 20). After taking a small aliquot from each sample (for subsequent protein content determination in Bradford assays), concentrated sample buffer (prewarmed at 95°C) was directly added to either cytoplasmic or nuclear fractions (to yield a final concentration of 1x sample buffer, i.e., 25 mM Tris base (pH 6.80), 2% SDS (w/v), 5% 2-ME (v/v), 10% glycerol, v/v), before a 3-min incubation at 95°C. All samples were electrophoresed on 10% denaturing gels prepared according to the method of Laemmli (47); equal loading was ascertained by adjusting sample volumes based on their respective protein content. After SDS-PAGE, proteins were transferred onto nitrocellulose membranes, which were stained with Ponceau Red, destained, and then processed for immunoblot analysis, as previously described (17).

Isolation of RNA protection assays and RPAs

Neutrophils were incubated in the presence or absence of stimuli or inhibitors for the desired times, as indicated. Total RNA was extracted according to a slightly modified Chomczynski and Sacchi procedure (48) and analyzed by RPA as previously described (49), using multiprobe templates from BD PharMingen (Mississauga, Ontario, Canada).

ELISA analysis of secreted proteins

Neutrophils (3 x 106 cells/600 µl) were cultured in 12-well culture plates at 37°C in 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 an in-house sandwich ELISA, using commercially available capture and detection Ab pairs (R&D Systems). Detection limits using these assays were 10 pg/ml or lower.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and distribution of Jun and Fos family proteins in resting and activated neutrophils

To determine which Jun/Fos family proteins are present in human neutrophils, unstimulated cells were boiled in sample buffer and processed for immunoblotting using specific Abs to Jun/Fos family members. Fig. 1A shows that substantial quantities of JunD and c-Fos are expressed in neutrophils, whereas the detection of a weak band comigrating with authentic c-Jun required that a sizable amount of material be loaded on the gels (i.e., at least 3 x 106 cells, as opposed to 0.5 x 106 cells for the other two proteins). A major immunoreactive band with an apparent molecular mass of ~60 kDa was also consistently detected (Fig. 1A), using two different c-Jun Abs. The scarcity of the c-Jun protein in neutrophils could not be explained by a rapid degradation through the ubiquitin/proteasome pathway (50), because an equally weak band was detected in neutrophils treated with proteasome inhibitors such as MG-132 or MG-262 (Fig. 2A and data not shown). By contrast, JunB, FosB, and Fra2 were not detected under any condition, even when up to 3 x 106 cell equivalents were loaded on the gels or when the films were deliberately overexposed (Fig. 1A and data not shown). Finally, Fig. 1B shows that well over one-half of the cellular levels of JunD and c-Fos were still detectable in neutrophils after a 2-h incubation in the presence of cycloheximide. This is somewhat less stable than the NF-{kappa}B/Rel proteins expressed in neutrophils (17) but far more stable than other related proteins, such as I{kappa}B-{alpha} (Ref. 17 and Fig. 1B). Thus, the Jun/Fos proteins appear to be of intermediate stability among transcriptional regulators expressed in neutrophils.



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  		FIGURE 1. Expression and stability of Jun/Fos proteins in human neutrophils (pmn). A, Freshly isolated neutrophils were boiled in sample buffer and processed for immunoblot analysis of their Jun/Fos proteins; 0.5 x 106 cells were loaded per lane, except for c-Jun (4 x 106 cells). B, Neutrophils were cultured for up to 2 h in the presence of 20 µg/ml cycloheximide (CHX). Cells were then boiled in sample buffer and processed for immunoblot analysis of their Jun/Fos proteins (24 µg/lane, representing ~0.8 x 106 cells/lane) and, for comparison, of I{kappa}B-{alpha}. Experiments are representative of at least three.

 


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  		FIGURE 2. Effect of neutrophil agonists on the cellular levels and distribution of Jun/Fos proteins. A, Neutrophils were cultured for 60 min in the presence or absence of a potent proteasome inhibitor (MG-132, 15 µM) and further stimulated with up to 1000 U/ml TNF-{alpha}, or 50 nM PMA, for up to 4 h. Cells were then boiled in sample buffer and processed for immunoblot analysis of their Jun/Fos protein content; for c-Fos and JunD 24 µg of protein were loaded per lane (~0.8 x 106 cell equivalents), whereas for c-Jun 90 µg of protein were loaded per lane (~3 x 106 cell equivalents). B, Neutrophils were incubated for 30 min in the presence or absence of up to 1000 U/ml TNF-{alpha} or 50 nM PMA. Cells were then disrupted by nitrogen cavitation, and the resulting cytoplasmic (Cyt) and nuclear (N) fractions were processed for immunoblot analysis of their Jun/Fos protein content (~106 cell equivalents/lane, representing 18 and 14 µg of protein per lane for cytoplasmic and nuclear fractions, respectively). We previously showed that such fractions are virtually devoid of cross-contamination (17 ). Experiments are representative of at least three. ctrl, Control.

 
Because previous studies had shown that mRNA species encoding several Jun/Fos family members rapidly accumulate in response to neutrophil stimulation with a variety of agonists (41, 42, 43, 44, 45), we examined whether this might result in increased amounts of the corresponding proteins. For this purpose, neutrophils were stimulated for various lengths of time (up to 4 h) with up to 1 µg/ml LPS, 1000 U/ml TNF-{alpha}, 100 nM fMLP, 50 nM PMA, or 1 nM GM-CSF, before immunoblot determination of their cellular content of Jun/Fos proteins. Somewhat surprisingly, the cellular levels of c-Jun, JunD, and c-Fos were largely unaffected by simulation with any of the above stimuli, even in the presence of MG-132, as shown in Fig. 2A in the case of TNF- and PMA-stimulated neutrophils. Similarly, JunB and FosB remained undetectable regardless of the stimulus used or of the incubation conditions, for up to 4 h (data not shown). Identical results were obtained whether neutrophils were incubated in polypropylene tubes in a waterbath with occasional agitation or cultured in TC-treated plasticware in an incubator. Finally, in response to PMA, a more slowly migrating c-Fos immunoreactive band was consistently detected (Fig. 2A), which presumably represents a phosphorylated isoform. The slower band was also observed in cells treated with MG-132 and TNF, albeit to a much lesser extent (Fig. 2A); moreover, the detection of this band required that the cells be stimulated for at least 25 min with either PMA or TNF (data not shown).

Although neutrophil activation entailed no significant change in the overall level of Jun/Fos proteins, we examined the possibility that the cellular distribution of Jun/Fos proteins might be affected after cell stimulation. To this end, subcellular fractions obtained by nitrogen cavitation of the cells were processed and analyzed by immunoblot; we previously showed that this fractionation procedure yields cytoplasmic fractions and intact nuclei that are exempt from cross-contamination (17, 20). Fig. 2C shows that both JunD and c-Fos are mostly cytoplasmic, although some nuclear c-Fos protein was always detectable. Cell stimulation with various neutrophil agonists did not significantly alter the cellular distribution of the Jun/Fos proteins, as shown for TNF and PMA in Fig. 2C. This said, it is noteworthy that the slowly migrating (presumably phosphorylated) c-Fos band was detected only in the nuclear fractions. Similar to the Jun/Fos proteins, JNK1 was mainly cytoplasmic (Fig. 3B), with weak amounts being detectable in the nuclear fractions (but only when the films were deliberately overexposed); neutrophil stimulation did not lead to an accumulation of nuclear JNK at any time point examined, up to 60 min (Fig. 3B and data not shown). Identical results were obtained in adherent neutrophils (data not shown).



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  		FIGURE 3. Effect of neutrophil agonists on AP-1 binding and JNK activity. A, Neutrophils were cultured for up to 60 min in the presence of up to 1000 U/ml TNF-{alpha}, 1 µg/ml LPS, or 3 nM GM-CSF. Cells were then disrupted by nitrogen cavitation, and nuclear extracts were prepared and analyzed in EMSA (6 µg/lane) using an AP-1 probe. For comparison, a nuclear extract from PMA-simulated PBMC was included on the gel (PBMC). B, Neutrophils were incubated for 30 min at 37°C in the presence or absence of either 1 µg/ml LPS or 50 nM PMA. Cells were then disrupted by nitrogen cavitation, and the resulting cytoplasmic (Cyt) and nuclear (N) fractions were processed for immunoblot analysis of their JNK1 content (~0.8 x 106 cell equivalents per lane, representing 14 and 12 µg of protein per lane for cytoplasmic and nuclear fractions, respectively). C, Freshly isolated PBMC were pretreated with a JNK inhibitor (SP 600125, 10 µM) for 30 min at 37°C and further incubated in the presence or absence of 50 nM PMA for 20 min. Cells were then boiled in sample buffer and processed for immunoblot analysis using anti-phospho-JNK Abs (40 µg/lane or ~1.5 x 106 cell equivalents). Experiments are representative of at least three. ctrl, Control.

 
Induction of an AP-1 DNA-binding activity and activation of JNK in human neutrophils

We next undertook to determine whether neutrophil activation by various stimuli results in the induction of AP-1 DNA-binding activity. Neutrophils were stimulated for varying lengths of time (up to 90 min) with fMLP, LPS, TNF-{alpha}, PMA, or GM-CSF before preparation of nuclear extracts and EMSA analysis. In 23 of 29 donors, a weak constitutive AP-1 DNA-binding activity was detectable in extracts from resting neutrophils (Fig. 3A), as we reported earlier (19). However, all of the agonists used failed to promote an increased binding to the AP-1 probe, as shown in Fig. 3A for TNF-, LPS-, and GM-CSF-stimulated neutrophils. In fact, somewhat decreased binding was even observed in some instances (12 of 29 donors). Under the same conditions, JNK1 neither translocated to the nucleus nor became phosphorylated (Fig. 3B and data not shown). By comparison, JNK phosphorylation readily took place in response to PMA in autologous PBMC (Fig. 3C).

In a recent study, JNK1 was reported to become phosphorylated in response to a high dose of TNF, under particular conditions (51). Briefly, neutrophils were resuspended at high density (20 x 106 cells/ml), and allowed to sediment for 55 min at 37°C in polypropylene tubes, before being diluted and plated in petri dishes for 25 min at 37°C and finally stimulated with TNF. Cells thus treated were referred to as adherent neutrophils, given that this protocol favored {beta}2 integrin engagement (51). We were able to reproduce these results by following the same procedure, although the extent of JNK activation (as assessed by its phosphorylation) was always modest, as shown in Fig. 4A. Under these conditions, the detection of phospho-JNK occurred only in response to TNF, because other known inducers of JNK activation such as LPS or even UV irradiation were mostly ineffective over a time course spanning up to 30 min (Fig. 4A and data not shown). The activation of JNK by TNF in these adherent neutrophils also did not lead to the detection of phospho-c-Jun, whereas TNF did promote the phosphorylation of endogenous c-Jun in autologous PBMC (Fig. 4B). However, our failure to detect phospho-c-Jun in neutrophils was to be expected, in view of the extremely low abundance of c-Jun in these cells. We therefore investigated whether AP-1 binding might be up-regulated under the same conditions leading to JNK activation in neutrophils. As shown in Fig. 5A, prior sedimentation of neutrophils, followed by plating and stimulation with a high dose of TNF, resulted in the induction of AP-1 DNA-binding activity. For comparison purposes, the same extracts were also analyzed in EMSA using a NF-{kappa}B probe (Fig. 5A, right); no differences were noted in the ability of TNF to induce NF-{kappa}B activation, regardless of whether neutrophils were adherent (i.e., plated with prior sedimentation) or not (i.e., incubated in polypropylene tubes). Similarly, no difference was observed whether freshly purified neutrophils were directly stimulated in suspension or plated in TC-treated plasticware and immediately stimulated. Finally, as in the case of phospho-JNK, an inducible AP-1 DNA-binding activity was detected only in neutrophils exposed to TNF; other stimuli such as LPS, GM-CSF, or fMLP were ineffective (data not shown).



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  		FIGURE 4. Activation of JNK in neutrophils submitted to sedimentation before stimulation (adherent neutrophils). A, Freshly isolated neutrophils were resuspended at high density (2 x 107 cells/ml) and allowed to sediment for 55 min at 37°C in polypropylene tubes; the cells were then diluted (to 3 x 106 cells/ml) and plated in TC-treated petri dishes for 25 min at 37°C. Cells were subsequently stimulated for up to 15 min with 1000 U/ml TNF-{alpha} or irradiated for 10 min with UV as described earlier (20 ). Cells were then boiled in sample buffer and processed for immunoblot analysis using anti-phospho-JNK Abs (60 µg/lane, representing ~2 x 106 cell equivalents). The membrane was subsequently reblotted using anti-p38 mitogen-activated protein kinase Abs. B, Neutrophils and autologous monocytes were incubated as described above, and whole cell samples were processed for immunoblot analysis using anti-phospho-c-Jun Abs. The experiments shown in this figure are representative of three.

 


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  		FIGURE 5. Induction and identification of an AP-1 DNA-binding activity in adherent neutrophils. A, Neutrophils were cultured under standard conditions (i.e., in TC-treated plasticware at 37°C in a 5% CO2 incubator; cultured only) or using an alternative protocol involving the sedimentation of the cells before culture (as described in the legend to Fig. 4A; adherent cells), and then stimulated with 1000 U/ml TNF-{alpha} or diluent for 15 min. Nuclear extracts (6 µg) were then prepared and analyzed in EMSA using an AP-1 probe (left) or a NF-{kappa}B probe (right); dried gels were exposed to film for 16 h (NF-{kappa}B) or for 3 days (AP-1). B, Neutrophils prepared following the sedimentation protocol were stimulated with 1000 U/ml TNF-{alpha}, and nuclear extracts (8 µg) were incubated in the presence of various Abs before the addition of labeled AP-1 probe and EMSA analysis. A 6-day autoradiograph is shown. Experiments are representative of at least four.

 
The detection of an increased AP-1 DNA-binding activity in response to TNF prompted us to characterize the nature of the inducible complex. As shown in Fig. 5B, supershift analysis using Abs to individual Jun/Fos proteins revealed that JunD and c-Fos Abs only displaced a minor proportion of the AP-1-binding complex, whereas Abs to c-Jun and JunB were ineffective. However, we found that an anti-CREB1 (but not an anti-CREB2) Ab consistently displaced the AP-1 complex (Fig. 5B). Similar results were obtained when using nuclear extracts from unstimulated adherent neutrophils or from nonadherent neutrophils (data not shown). Because our anti-CREB1 Ab cross-reacts with the ATF1 transcription factor (according to the supplier’s technical literature), we also ascertained whether anti-ATF1 Abs might exert a similar effect, which they did not (Fig. 5B). Immunoblot analyses confirmed that both CREB1 and CREB2 are expressed in neutrophils (data not shown).

Consequences of JNK pathway inhibition in human neutrophils

Although our data indicated that AP-1 is an unlikely participant in granulocyte transcriptional activation, the occurrence of JNK activation in sedimented neutrophils nonetheless made it conceivable that JNK might still somehow have an impact on transcriptional events leading to gene expression in these cells. To clarify this point, we investigated the functional consequences of blocking the JNK signaling cascade in neutrophils. To this end, neutrophils were pretreated with a recently available pharmacological inhibitor of JNK, SP 600125 (52), before stimulation with either TNF or LPS, and subsequent assessment of cytokine gene expression by RPA. In agreement with previous studies, steady-state levels of mRNA transcripts encoding IL-8, Mip-1{alpha}, Mip-1{beta}, and TNF-{alpha} were much enhanced in stimulated neutrophils, relative to resting cells (Fig. 6). Pretreatment with the JNK inhibitor only marginally altered inflammatory cytokine gene expression, in both resting and activated cells, and whether the cells had been submitted to prior sedimentation or not (Fig. 6 and data not shown). In keeping with these findings, the synthesis and release of the corresponding cytokines in LPS- or TNF-activated neutrophils were not subject to inhibition by SP 600125, be it in adherent or nonadherent cultured cells (Fig. 7), with the single exception of Mip-1{beta} synthesis (but not secretion) in TNF-treated adherent neutrophils.



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  		FIGURE 6. Effect of JNK inhibition on inflammatory cytokine expression in neutrophils. Cells were cultured in the presence ({blacksquare}) or absence () of a highly selective JNK inhibitor (20 µM SP 600125) for 30 min and stimulated for 60 min with up to 1 µg/ml LPS or 1000 U/ml TNF-{alpha}. Total RNA was extracted and analyzed in RPA; individual mRNA species were quantitated and standardized using GAPDH. Mean ± SD of three independent experiments, except for TNF-{alpha} mRNA (two experiments).

 


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  		FIGURE 7. Effect of JNK inhibition on inflammatory cytokine synthesis and release by neutrophils (pmn). Neutrophils were either made adherent or not, before being cultured for 30 min in the presence or absence of the highly selective JNK inhibitor, SP 600125 (20 µM), and subsequently stimulated with up to 1 µg/ml LPS or 1000 U/ml TNF-{alpha} for 2 h. The inflammatory cytokine content of the resulting culture supernatants and of the corresponding cell pellets was then assessed by ELISA. Mean ± SD of at least three independent experiments. {blacksquare}, Cytokine release; , total cytokine synthesis (i.e., cell-associated plus secreted). ctrl, Control.

 
Expression of JNK- or AP-1-interacting factors in human neutrophils

To further explore the reasons behind the weak inducibility of the JNK/AP-1 cascade in neutrophils, we investigated whether some of the known factors interacting with JNK or with the Jun/Fos proteins are expressed in neutrophils. One such factor is JNK-interacting protein (JIP-1), which was initially proposed to retain JNK in the cytosol (53). When neutrophils were examined for its presence, however, none could be detected by immunoblot (data not shown), although we had difficulty finding a cell line that could serve as a positive control. Redox factor-1 (Ref-1) is another such factor. It is a nuclear protein activated by thioredoxin, which keeps certain cysteine residues on the Jun/Fos proteins in a reduced state, thereby preserving their ability to bind DNA (54, 55, 56, 57). Immunoblot experiments demonstrated that Ref-1 is readily detectable in neutrophils and that its abundance is unaffected after stimulation of the cells with TNF-{alpha}, LPS, or PMA for up to 2 h, be it in cultured cells or in adherent neutrophils (Fig. 8A and data not shown).



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  		FIGURE 8. Expression of Ref-1 in primary neutrophils and during granulocytic differentiation of a human promyeloid cell line. A, Neutrophils were cultured for 2 h in the presence of 100 U/ml TNF-{alpha}, 100 ng/ml LPS, or 50 nM PMA. Cells were then boiled in sample buffer before immunoblot determination of their Ref-1 content (16 µg of protein per lane or ~0.5 x 106 cell equivalents). B, PLB-985 cells were used either undifferentiated (Undiff) or after 4 days of culture in the presence of 1.25% DMSO; granulocytic differentiation was ascertained by monitoring the appearance of surface markers CD11b and CD11c, as well as by the acquisition of fMLP responsiveness (not shown). Undifferentiated or granulocytic PLB-985 cells were stimulated for 15 min with 50 nM PMA or irradiated with UV for 10 min. The cells were then boiled in sample buffer and processed for immunoblot analysis using Abs to Ref-1 (15 µg of protein per lane or ~0.4 x 106 cell equivalents), phospho-JNK (60 µg of protein of lane), or JNK1 (20 µg of protein per lane). Experiments are representative of three. ctrl, Control.

 
Because it was recently reported that the in vitro differentiation of peripheral monocytes into macrophages led to diminished Ref-1 protein levels and a markedly decreased ability to activate AP-1 (58), we also investigated whether a similar process might take place during granulocytic differentiation. For this purpose, promyelocytic PLB-985 cells were differentiated with DMSO into neutrophil-like cells, and Ref-1 levels were examined by immunoblot. As shown in Fig. 8B, the cellular amount of Ref-1 was only slightly diminished in granulocytic-differentiated PLB-985 cells, relative to undifferentiated cells. In sharp contrast, granulocytic differentiation also resulted in a much decreased ability of the cells to activate JNK in response to UV irradiation or PMA (Fig. 8B), whereas JNK1 levels were mostly unaffected by granulocytic differentiation (Fig. 8B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study, we set out to investigate the role of the JNK/AP-1 signaling pathway in inflammatory cytokine production by human neutrophils. We first determined that among Jun/Fos proteins, only c-Fos and JunD are abundantly expressed in neutrophils; additionally, very low levels of the c-Jun protein seem to be present. These proteins were mostly cytoplasmic in both resting and activated neutrophils; additionally, some c-Fos (but not JunD) was consistently detected in nuclear fractions. Unexpectedly, the cellular levels of these proteins were unaffected by neutrophil stimulation with a series of agonists that cause a rapid accumulation of mRNA transcripts encoding various Jun/Fos family members in these cells. This could not be attributed to a rapid degradation of the proteins, because identical results were obtained in the presence of proteasome inhibitors, which prevent the targeted degradation of at least c-Jun and c-Fos (50, 59). Moreover, cycloheximide experiments demonstrated that among transcriptional regulators known to be expressed in neutrophils, the Jun/Fos proteins are relatively stable, with a half-life far exceeding 2 h. Taken together, these observations suggest that the Jun/Fos proteins are inefficiently translated in neutrophils, something that might contribute to their relatively slow turnover rate. Consistent with this interpretation is that although neutrophils constitutively express much higher levels of c-Fos, c-Jun, and JunD mRNA than monocytes (40, 41, 42, 45, 60), the corresponding proteins are generally more abundant in PBMC than in autologous neutrophils (our unpublished data); this is especially obvious in the case of c-Jun.

Another marked difference between PBMC and neutrophils is the apparent inability of the latter to activate the AP-1 transcription factor. We had previously reported that a basal AP-1 signal could be detected in unstimulated cells but that no AP-1 activation occurred in response to IL-15 or IL-2 stimulation of neutrophils, contrary to autologous PBMC (19). We now show that under various incubation conditions, and in response to a number of potent neutrophil stimuli, no increased binding to a canonical AP-1 element is detected. In fact, the single experimental procedure that yielded an enhanced AP-1 binding in EMSA (i.e., adherence) was the same that allows the detection of JNK phosphorylation in these cells (Ref. 51 and this paper). Whether the two processes are linked is doubtful, especially because the enhanced AP-1 binding appeared to result in a good part from a lower constitutive binding to the AP-1 probe in adherent cells (Fig. 5A). Regardless, the nature of this inducible complex made it clear that it does not correspond to a typical AP-1 dimer. Indeed, we found that a major component of this DNA-binding activity is CREB1, whereas c-Fos appeared to be a minor component at best. Because Jun/Fos proteins are not known to associate with CREB1, a binding partner for the latter remains to be identified. In this regard, it has been reported that members of the Jun, Fos, and CREB families can heterodimerize with certain ATF family proteins (61), and although ATF-1 does not seem to be involved based on our supershift data, other ATF family members might be. Alternatively, CREB1 might bind to the AP-1 element as a homodimer. Likewise, potential binding partners for c-Fos (which cannot homodimerize) have eluded identification. Although it has been reported that RelA, which is also expressed in neutrophils (17), can associate with c-Fos (30), Abs raised against RelA, c-Rel, and p50/NF-{kappa}B1 failed to affect the detection or electrophoretic migration of the inducible AP-1 complex (our unpublished data). In summary, neutrophils exhibit a constitutive AP-1-binding activity that can be modulated only under very specific conditions but that does not correspond to a true AP-1 complex. In a recent study, Page et al. (62) similarly detected a constitutive binding to an AP-1 probe in neutrophils. Contrary to the present findings, however, they observed a moderate increase in AP-1 binding when neutrophil suspensions were stimulated for 60 min (but not 20 min) with 1 µg/ml LPS (62). Because no supershifts were performed, it is difficult to determine whether the DNA-binding activity was made up of AP-1 dimers or of other constituents. In view of the data presented herein and of the fact that their inducible AP-1 activity was partially displaced by a nonspecific competitor (62), it would appear that the observed complex was probably not genuine AP-1, in agreement with our conclusions.

In keeping with their inability to activate AP-1, neutrophils also failed to activate JNK under most experimental conditions. Before our study, a number of investigators had indeed reported that JNK does not become activated after neutrophil exposure to such diverse stimuli as cytokines and growth factors including: TNF-{alpha}, IL-1{beta}, GM-CSF, and G-CSF (63, 64, 65); LPS (66); phorbol esters (67); chemotactic factors including PAF, fMLP, and IL-8 (67, 68, 69, 70); opsonized phagocytic particles (71); or even potent JNK activators such as UV irradiation, heat shock, chemical stress, or hypertonicity (72, 73). In the current study, we confirmed that JNK activation is undetectable in neutrophils under many of the above conditions, albeit with one notable exception. If the cells are first sedimented at high density to promote {beta}2 integrin engagement, then JNK can become phosphorylated in response to a high dose of TNF, as recently reported by another group (51). Even so, it must be stressed that JNK activation occurs on a modest scale under these conditions and that other stimuli which we tested in adherent neutrophils (such as LPS, fMLP, and UV irradiation) failed to similarly activate JNK. More importantly, JNK activation appears to be of little consequence to downstream responses that are normally under its influence. As exposed above, no experimental condition, whether it led to JNK activation or not, resulted in the activation of genuine AP-1 complexes in neutrophils. Accordingly, we showed that a potent JNK inhibitor (SP 600125) failed to inhibit inflammatory cytokine gene expression, synthesis, and secretion in neutrophils under all conditions tested. By comparison, the same concentration of SP 600125 used herein (20 µM) blocked cyclooxygenase-2 and TNF-{alpha} gene expression in human PBMC and purified monocytes stimulated with LPS (52). Overall, our data make it unlikely that in human neutrophils, JNK plays any significant role in AP-1 activation or inflammatory cytokine production. Whether JNK activation has an impact on other neutrophil responses or cellular processes remains unclear at this stage. Avdi et al. (51) recently proposed a link between JNK activation and the onset of apoptosis in adherent neutrophils exposed to TNF, based on the observation that anti-CD11b Abs could attenuate both processes, as well as the activation of the kinase cascade upstream of JNK. However, it is also possible that the induction of apoptosis as well as the mobilization of the kinase cascade leading to JNK activation represent two parallel processes resulting from the combined engagement of {beta}2 integrins and TNF receptors. Thus, a role for JNK in neutrophil activation still awaits a formal demonstration.

There are several potential explanations for the weak propensity of neutrophils to activate AP-1 and JNK. First, the very low levels of c-Jun are likely to limit the formation of any prototypical AP-1 to all but insignificant amounts. Moreover, any phosphorylation of c-Jun (or more likely, JunD) within these complexes would occur only if the exacting requirements for JNK activation are met. And even so, JunD remained strictly cytoplasmic under all conditions tested. Similarly, only weak amounts of JNK were localized to the nucleus, but these levels were not increased after stimulation. This being said, JNK itself, as well as the upstream kinases (including MKK4/MKK7 and MEKK1), do not seem to be dysfunctional, because the entire signaling cascade is activatable, at least under conditions favoring {beta}2 integrin engagement before TNF stimulation (51). However, it is also quite clear that the JNK signaling pathway functions less efficiently in neutrophils than in other inflammatory cells. In this regard, it is noteworthy that the ability to activate JNK is gradually lost during the granulocytic differentiation of the human promyeloid cell lines, HL-60 (72) and PLB-985 (this study). Granulocytic differentiation of HL-60 cells was also reported to result in a substantially reduced ability to activate AP-1 (74), consistent with our neutrophil data. Interestingly, a similar loss of AP-1 inducibility was observed when human monocytes are differentiated into macrophages (58), and AP-1 activation is also impaired in terminally differentiated human alveolar macrophages (75). Low levels of the Ref-1 protein were proposed as a reason for this functional impairment in macrophages (58). Although Ref-1 was readily detectable in neutrophils, its cellular levels may nonetheless be limiting; alternatively, it may remain unphosphorylated, which would compromise its function (76). Either scenario would have far reaching consequences, because AP-1 activity is regulated in good part by the redox status of certain cysteine residues in its constituent subunits (77) and because JNK activity is reportedly sensitive to redox regulation as well (78). In addition to Ref-1, several other cellular factors (either individually or in concert) might also contribute to hindering the efficiency of the JNK/AP-1 system in mature neutrophils. One such factor, JIP-1, is increasingly viewed as an important scaffolding protein that may participate in the assembly of a functional JNK signaling complex (79, 80). Thus, the apparent lack of JIP-1 in neutrophils could contribute to the difficulties encountered in activating JNK. Another potentially important cellular factor is JAB-1 (Jun activation domain-binding protein-1), which was found to stabilize binding of both Jun and JunD to AP-1 sites, thereby enhancing c-Jun- and JunD-driven transcription (81). Whether JAB1 is present in neutrophils remains to be determined. Finally, a potentially crucial factor might be the activity of protein phosphatases that control JNK activity, such as the M3/6 phosphatase (82) or the recently characterized MKP7 (mitogen-activated protein kinase kinase phosphatase-7) (83, 84).

Although the reasons for which the JNK/AP-1 signaling pathway behaves so differently in neutrophils (relative to most other circulating leukocytes) require further elucidation, our data strongly suggest that this pathway has little or no impact on the expression of inflammatory mediators in neutrophils, at least under the various conditions tested here. This is particularly interesting for cytokines such as TNF-{alpha}, IL-1{beta}, IL-1RA, IL-8, and others, the promoters of which have been described as being at least partially dependent on AP-1 (21, 22, 23, 27), in that it shows that AP-1 is clearly not essential for their induction. Conversely, the human MCP-1 gene promoter is known to be dependent on both NF-{kappa}B and AP-1 for inducibility (28), and in view of the present findings, it is probably not coincidental that most studies having addressed the issue of MCP-1 production by neutrophils determined that it does not take place. On the one hand, MCP-1 mRNA and protein were readily detected after 20 h in culture (but not in fresh neutrophils) by one group (85), whereas another group reported that their detection was observed after an overnight incubation of neutrophils with supernatants from PHA-stimulated PBMC (86). By contrast, no MCP-1 gene or protein was detected in neutrophils by several other investigators under a rather exhaustive array of conditions, be it in culture or after adhesion to a substrate, or in cells exposed to a variety of stimuli (either alone or in combination), including LPS, TNF-{alpha}, IL-1{beta}, IFN-{beta}, IFN-{gamma}, GM-CSF, fMLP, leukotriene B4, PMA, phagocytic stimuli, immobilized anti-ICAM Abs, fibrinogen, or inflammatory microcrystals, for up to 48 h (8, 87, 88, 89, 90, 91). Similarly, we never detected any MCP-1 signal in our numerous RPA analyses of neutrophil RNA preparations, whereas MCP-1 mRNA is readily apparent in RNA samples from activated PBMC (our unpublished data). Thus, although it cannot be excluded that neutrophils might produce some MCP-1 under very specific conditions, it can safely be stated that under most conditions they do not. This therefore lends further support to our contention that the JNK/AP-1 pathway has little if any impact on inflammatory mediator expression in neutrophils.


   Footnotes
 
1 This work was supported by Canadian Institutes for Health Research Grants MOP 36349 and MOP 62705, Canadian Foundation for Innovation Grant 3946, and Gouvernement du Québec (to P.P.M.). P.P.M. is a Scholar of the Medical Research Council of Canada. Back

2 E.C. and T.E. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Patrick P. McDonald, Centre de Recherche Clinique, 3001, 12e avenue Nord, pièce 4849, Sherbrooke, Québec, Canada J1H 5N4. E-mail address: patrick.mcdonald{at}USherbrooke.ca Back

4 Abbreviations used in this paper: Mip, macrophage-inflammatory protein; MCP, monocyte chemoattractant protein; ATF, activating transcription factor; JNK, c-Jun N-terminal kinase; DFP, diisopropyl fluorophosphate; RPA, RNase protection assay; JIP-1, JNK-interacting protein; Ref-1, redox factor-1; TC, tissue culture. Back

Received for publication October 28, 2002. Accepted for publication July 23, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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