NF-kappa B Activation in Tumor Necrosis Factor alpha -stimulated Neutrophils Is Mediated by Protein Kinase Cdelta

CORRELATION TO NUCLEAR Ikappa Balpha *

Ivana VancurovaDagger, Veronika Miskolci, and Dennis Davidson

From the Division of Neonatal-Perinatal Medicine, Schneider Children's Hospital, Long Island Jewish Medical Center-The Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, New York 11040

Received for publication, January 10, 2001, and in revised form, March 6, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The transcription factor NF-kappa B is critical for the expression of multiple genes involved in inflammatory responses and apoptosis. However, the signal transduction pathways regulating NF-kappa B activation in human neutrophils in response to stimulation with tumor necrosis factor-alpha (TNFalpha ) are undefined. Since recent studies implicated activation of NF-kappa B as well as protein kinase C-delta (PKCdelta ) in neutrophil apoptosis, we investigated involvement of PKCdelta in the activation of NF-kappa B in TNFalpha -stimulated neutrophils. Specific inhibition of PKCdelta by rottlerin prevented Ikappa Balpha degradation and NF-kappa B activation in TNFalpha -stimulated neutrophils. This regulation of NF-kappa B activation by PKCdelta was specific only for TNFalpha signaling, since lipopolysaccharide- or interleukin-1beta -induced NF-kappa B activation and Ikappa Balpha degradation were not inhibited by rottlerin. In addition, we show that in human neutrophils, but not monocytes, Ikappa Balpha localizes in significant amounts in the nucleus of unstimulated cells, and the amount of Ikappa Balpha in the nucleus, as well as in the cytoplasm, correlates with the NF-kappa B DNA binding. These results suggest that in human neutrophils, the presence of Ikappa Balpha in the nucleus may function as a safeguard against initiation of NF-kappa B dependent transcription of pro-inflammatory and anti-apoptotic genes, and represents a distinct and novel mechanism of NF-kappa B regulation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neutrophils (polymorphonuclear leukocytes) are short-lived terminally differentiated blood cells that play a vital role in the inflammatory response; they are one of the first cells recruited to the site of injury or infection (1, 2). In addition to their phagocytic and killing properties, neutrophils synthesize numerous proinflammatory cytokines and chemokines, including TNFalpha ,1 interleukin (IL)-1alpha and IL-1beta , IL-8, and macrophage inflammatory protein alpha , that may amplify the inflammatory process (3-8). Expression of many of these proinflammatory proteins is regulated at the level of gene transcription by transcription factor NF-kappa B (9, 10).

Since the knowledge that neutrophils are an important source of cytokines is relatively new, the molecular mechanisms regulating cytokine expression in these cells have only begun to be investigated (11, 12). We have previously shown that NF-kappa B activity in human neutrophils consists of p50/50 homodimers and p50/65 heterodimers, and that their activation in TNFalpha -stimulated neutrophils is inhibited by dexamethasone, an anti-inflammatory drug (13). Using pharmacological inhibitors Nick et al. (12) demonstrated that lipopolysaccharide (LPS)-induced activation of NF-kappa B in neutrophils is mediated by p38alpha mitogen-activated protein kinase. However, the signaling pathways leading to NF-kappa B activation in response to neutrophil stimulation with TNFalpha are undefined.

Interestingly, a recent study demonstrated that NF-kappa B also regulates both constitutive and TNFalpha -induced apoptosis in human neutrophils (14). This neutrophil apoptosis has been recently shown to be mediated by a novel isoform of protein kinase C (PKC), PKCdelta (15, 16). Therefore, we sought to investigate involvement of PKCdelta in TNFalpha -induced activation of NF-kappa B in human neutrophils. PKCdelta is selectively inhibited by rottlerin (15, 17-19), and as any other novel PKC, it is activated in a Ca2+-independent manner by diacylglycerol (DAG), which is produced by activated phospholipase C (PLC) (20).

In this study, we show that inhibition of phosphatidylinositol-specific phospholipase C (PI-PLC) and PKCdelta blocks activation of NF-kappa B in TNFalpha -stimulated human neutrophils by inhibiting degradation of Ikappa Balpha . The regulation of NF-kappa B activation by PKCdelta is specific only for TNFalpha signaling, since LPS- or IL-1beta -induced activation of NF-kappa B and degradation of Ikappa Balpha are not inhibited by rottlerin. In addition, we show that in human neutrophils, but not monocytes, Ikappa Balpha localizes in significant amounts in the nucleus of resting unstimulated cells. The NF-kappa B DNA binding in the neutrophil does not correlate with nuclear translocation of NF-kappa B subunits, as is the case in most mammalian cells (21-23), but rather with the amount of Ikappa Balpha in the nucleus, as well as in the cytoplasm.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Ficoll-Paque PLUS, dextran T-500, T4 polynucleotide kinase, poly(dI-dC), and Sephadex G25 spin columns were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Hank's balanced salt solution, RPMI 1640 medium, and endotoxin tested, heat-inactivated fetal calf serum (FCS) were obtained from Life Technologies (Grand Island, NY). Escherichia coli expressed purified recombinant human TNFalpha and IL-1beta were purchased from R & D Systems (Minneapolis, MN). [32P]ATP was purchased from PerkinElmer Life Sciences (Boston, MA). Histone H1, U-73122, U-73343, D-609, Et-18-OCH3, rottlerin, and Ro-31-8425 were purchased from Calbiochem (La Jolla, CA). Protein A/G Plus-agarose, purified polyclonal antibodies to human p50 (sc-7178X), Ikappa Balpha (sc-371), PKCdelta (sc-937), and glucocorticoid receptor (GR, sc-1003), and mouse monoclonal anti-actin antibody (sc-8432) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibody to p65 (SA-171) was obtained from Biomol (Plymouth Meeting, PA). Monoclonal mouse anti-SUMO-1 antibody was from Zymed Laboratories Inc. (San Francisco, CA), and polyclonal lactate dehydrogenase (LDH) antibody (20-LG22) was purchased from Fitzgerald Industries International (Concord, MA). Horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and anti-goat IgG secondary antibodies were from Amersham Pharmacia Biotech (Arlington Heights, IL). All other reagents were molecular biology grade and were purchased from Sigma. All reagents and plasticware used throughout the experiments were pyrogen-free.

Cell Isolation and Culture-- Fresh blood was obtained from healthy adult human volunteers and collected in heparinized preservative-free tubes. Neutrophils and monocytes (95-98% purity) were separated under endotoxin-free conditions using Ficoll-Paque centrifugation (24), and the neutrophils were subsequently purified by dextran sedimentation and hypotonic lysis of residual erythrocytes as described previously (6). Purified cells were resuspended in RPMI 1640 supplemented with 5% low endotoxin fetal calf serum, at a final concentration of 5 × 106 cells/ml, and incubated at 37 °C in polypropylene tubes with gentle agitation. For the inhibition experiments, the inhibitors were dissolved in dimethyl sulfoxide, and the cells were pretreated 15 min with either the inhibitor or with Me2SO alone, before stimulation with TNFalpha . The incubations were terminated by placing cells on ice and rapid centrifugation (1 min, 5,000 × g, 4 °C).

Preparation of Cytoplasmic and Nuclear Extracts-- Nuclear and cytoplasmic extracts were prepared from 5 × 106 cells as described previously (13). Briefly, the pelleted cells were resuspended in 300 µl of hypotonic buffer (buffer A: 10 mM Hepes, pH 7.5, 10 mM KCl, 3 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 2 mM dithiothreitol) containing the following protease and phosphatase inhibitors: 2 mM phenylmethylsulfonyl fluoride, 100 µg/ml soybean trypsin inhibitor, 1 mM benzamidine, 2 mM levamisole, 1 mM Na3VO4, 10 mM NaF, 20 mM glycerophosphate, and protease inhibitor mixture from Sigma (P-8340), used at concentration 60 µl/5 × 106 cells). After 15 min incubation on ice, 0.05 volumes of 10% Nonidet P-40 were added, the cells were vortexed (10 s) and immediately centrifuged at 500 × g for 10 min at 4 °C. The supernatants were collected, designated as cytoplasmic extracts, aliquoted, and stored at -80 °C.

The nuclear pellets were washed in 200 µl of buffer A containing the protease inhibitors, and re-centrifuged. The pelleted nuclei were resuspended in 50 µl of ice-cold nuclear buffer (NE buffer: 20 mM Hepes, pH 7.5, 25% glycerol, 0.8 M KCl, 1 mM MgCl2, 1% Nonidet P-40, 0.5 mM EDTA, 2 mM dithiothreitol) containing the protease and phosphatase inhibitors as described above. Following a 20-min incubation on ice (with occasional mixing), the samples were centrifuged (14,000 × g, 15 min, 4 °C), and the resulting supernatants (nuclear extracts) were aliquoted and stored at -80 °C. Protein concentration was measured using the Pierce Coomassie Plus protein assay kit (Pierce, Rockford, IL). Contamination of nuclear and cytoplasmic fractions by cytoplasmic and nuclear proteins, respectively, was determined by Western analysis using LDH and SUMO-1 as specific markers.

Electrophoretic Mobility Shift Assay (EMSA)-- The oligonucleotide used as a probe for EMSA was a 42-base pair double-stranded construct (5'-TTGTTACAAGGGGACTTTCCGCTGGGGACTTTCCAGGGAGGC-3') containing two tandemly repeated NF-kappa B-binding sites (underlined). Mutant oligonucleotide used for competition studies was 5'-TTGTTACAATCTCACTTTCCGCTTCTCACTTTCCAGGGAGGC-3'. End labeling was accomplished by treatment with T4 kinase in the presence of [gamma -32P]ATP, and the labeled oligonucleotide was purified on a Sephadex G-25 column, as described elsewhere (25).

Nuclear extracts (containing 4-6 µg of protein in 5-7 µl) were incubated (20 min at room temperature) with 5-10 fmol of radiolabeled oligonucleotide (~70,000 cpm) in 20 µl of binding buffer (20 mM Tris-Cl, pH 7.5, 150 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, 6% glycerol) supplemented with 20 µg of acetylated bovine serum albumin and 2 µg of poly(dI-dC). For competition or supershift experiments, binding reactions were performed in the presence of 30 M excess of unlabeled oligonucleotide or 1 µg of specific polyclonal antibody, respectively, and incubated 15 min at room temperature before adding 32P-labeled oligonucleotide. The resulting complexes were resolved on 5% nondenaturing polyacrylamide gels that had been pre-run at 100 V for 30 min in 0.5 × TBE buffer. Electrophoresis was conducted at 180 V for 2.5 h. After electrophoresis, gels were transferred to Whatman DE-81 paper, dried, and exposed to autoradiographic film (Kodak BioMax MS) with intensifier screen at -80 °C.

Immunoprecipitation PKCdelta Assay-- PKCdelta enzymatic activity was assayed in whole cell lysates immunoprecipitated by PKCdelta specific polyclonal antibody as follows. Neutrophils (5 × 106) were lysed in 0.3 ml of lysis buffer (50 mM Tris-Cl, pH 8.0, 250 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 2 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 100 µg/ml soybean trypsin inhibitor; 1 mM benzamidine, 2 mM levamisole, 1 mM Na3VO4, 20 mM glycerophosphate, 10 mM NaF and protease inhibitor mixture from Sigma (P-8340), used at concentration 60 µl/5 × 106 cells). Soluble proteins were pre-cleared by a 1-h incubation (4 °C) with 10 µl of Protein A/G Plus-agarose. The precleared supernatants were incubated with 1 µg of anti-PKC-delta or control anti-GR antibody (2 h, 4 °C), and immunoprecipitated with 10 µl of Protein A/G Plus-agarose for an additional 1 h. The immune complexes were washed 5 times with lysis buffer and 1 time with kinase buffer (20 mM Hepes, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 20 µM ATP), and resuspended in 20 µl of kinase buffer. Five µl of 5 × reaction buffer (1 mg/ml histone H1, 20 µM 1,2-dioleoyl-sn-glycerol, and 0.25 mg/ml L-alpha -phosphatidyl-L-serine) and 5 µCi of [gamma -32P]ATP were added, and the samples were incubated for 5 min at 30 °C. Reactions were stopped by the addition of 8 µl of 5 × sample buffer, the samples were boiled and resolved on a 12% SDS-polyacrylamide gel. The gels were stained with Coomassie, and the extent of histone H1 phosphorylation was determined by both autoradiography and scintillation counting of the excised Coomassie-stained histone polypeptide bands. In experiments examining the effect of rottlerin on PKCdelta activity in vitro, rottlerin was added to the PKCdelta immunoprecipitates in concentrations given in the text before the addition of 1 µM ATP.

Western Blotting-- Denatured proteins were separated on 12% denaturing polyacrylamide gels and transferred to nitrocellulose membrane (Hybond C; Amersham Pharmacia Biotech). Membranes were blocked overnight with a 5% (w/v) nonfat dry milk solution containing 10 mM Tris-Cl, pH 7.5, 140 mM NaCl, 1.5 mM MgCl2, and 0.1% Tween 20 (TBSTM) before incubating with primary antibodies (1 h for Ikappa Balpha , p65, p50, SUMO-1, and LDH antibodies, and overnight for actin antibody). Primary antibodies were diluted in TBSTM (1:250 for Ikappa Balpha and actin, 1:700 for p65, 1:300 for p50, and 1:200 for LDH and SUMO-1). After washing, the membranes were incubated 1 h with horseradish peroxidase-labeled secondary antibody diluted 1:2000 in TBSTM, and the labeled proteins were detected using enhanced chemiluminescence (ECL) reagents as described by the manufacturer (Amersham Pharmacia Biotech).

To confirm equivalent amounts of loaded proteins, or to re-probe the membrane with another antibody, the membranes were stripped with 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-Cl (pH 6.7) for 30 min at 50 °C, and incubated with the appropriate primary antibody diluted in TBSTM. The signal was developed using secondary IgG-horseradish peroxidase and ECL detection as described above.

Data Analysis-- Data presented here represent a minimum of three experiments, and, where appropriate, data are expressed as mean ± S.E. Statistical significance was evaluated by using ANOVA.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Specific Inhibitors of PI-PLC and PKCdelta Block Activation of NF-kappa B in TNFalpha -stimulated Neutrophils-- To investigate whether the TNFalpha -induced NF-kappa B activation involves PLC- and PKCdelta -dependent pathways, we used inhibitors of phosphatidylcholine (PC)- and phosphatidylinositol (PI)-specific PLC, and PKCdelta : D-609 (26), U-73122, and Et-18-OCH3 (27-29), and rottlerin (15, 17-19), respectively. Neutrophils were preincubated 15 min with or without the corresponding inhibitor, stimulated 30 min with TNFalpha , and the NF-kappa B DNA binding activity was measured in nuclear extracts by EMSA. As seen in Fig. 1A, neutrophil stimulation with TNFalpha induced activation of the p50/65 heterodimer, and to a lower extent also the p50/50 homodimer. The specificity and identity of these complexes was confirmed using competition and supershift assay as shown in panel B. Since the NF-kappa B form responsible for induction of inflammatory and apoptotic genes is the p50/65 heterodimer, whereas the cellular function of the p50/50 homodimer is not fully understood (30), we focused on DNA binding activity of the p50/65 NF-kappa B heterodimer.


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Fig. 1.   The PI-PLC and PKCdelta specific inhibitors abolish TNFalpha -induced NF-kappa B activation in human neutrophils. A, neutrophils were preincubated 15 min with U-73122 (5 µM), D-609 (50 µM), Et-18-OCH3 (50 µM), and rottlerin (50 µM), and then stimulated with TNFalpha (10 ng/ml) for 30 min. NF-kappa B DNA binding was measured by EMSA in nuclear extracts. The result is representative of three separate experiments. B, competition and supershift analysis of NF-kappa B DNA binding complexes in nuclear extracts from TNFalpha -stimulated human neutrophils. Nuclear extracts were incubated with 32P-labeled NF-kappa B specific DNA probe alone (lane 1) or in the presence (+) of 30 M excess of unlabeled wild type (wt) NF-kappa B oligonucleotide (lane 2), or a mutant oligonucleotide (lane 3). Antibodies used in supershift assay included antibodies to p50 (lane 4) and p65 (lane 5). This experiment is representative of four.

The p50/65 NF-kappa B DNA binding activity was inhibited by U-73122 (5 µM) and Et-18-OCH3 (50 µM), inhibitors of PI-PLC, and by PKCdelta inhibitor rottlerin (50 µM). In contrast, inhibitor of PC-PLC, D-609 at 50 µM concentration previously shown to be selectively effective to inhibit PC-PLC activity (26, 31) did not reduce NF-kappa B DNA binding (Fig. 1A). The inhibition of NF-kappa B DNA binding by U-73122 was dose dependent (Fig. 2A). The complete inhibition of p50/65 NF-kappa B was achieved at 5 µM U-73122 concentration, and the IC50 was ~2 µM. This IC50 value is consistent with the previously reported IC50 for PLC specific inhibition by U-73122 in the neutrophil (27, 28). The inactive structural analogue of U-73122, U-73343, in the range of 0.1-5 µM concentrations, had no effect on NF-kappa B DNA binding (data not shown).


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Fig. 2.   U-73122 and rottlerin inhibit TNFalpha -induced activation of NF-kappa B in human neutrophils in a dose-dependent manner. A, autoradiography showing dose response of U-73122 on TNFalpha -induced NF-kappa B activation. Neutrophils were preincubated with varying concentrations of U-73122 for 15 min, and stimulated with TNFalpha (10 ng/ml) for 30 min. NF-kappa B DNA binding was measured in nuclear extracts by EMSA. B, autoradiograph showing dose response of rottlerin on TNFalpha -induced NF-kappa B activation. Neutrophils were preincubated with varying concentrations of rottlerin for 15 min, and stimulated with TNFalpha (10 ng/ml) for 30 min. Both autoradiographs are representative of three experiments.

Fig. 2B shows a dose response of the PKCdelta -specific inhibitor rottlerin on NF-kappa B DNA binding in TNFalpha -stimulated neutrophils. The complete inhibition of the p50/65 NF-kappa B heterodimer was achieved by rottlerin concentrations of 50 µM, and the IC50 was ~10 µM. These values correlate well with the previously reported rottlerin IC50 for PKCdelta inhibition 3-6 µM, whereas the IC50 values for other PKC isoforms were 40-100 µM (17-19). In contrast, neutrophil pretreatment with Ro-31-8425 (100 nM), which inhibits the classical isoforms of PKC but not PKCdelta (32), had no inhibitory effect on TNFalpha -induced NF-kappa B DNA binding even at concentration 10 times higher than the reported IC50 (data not shown). Importantly, the inhibitory effect of rottlerin on NF-kappa B DNA binding was specific only for TNFalpha induction, since LPS, as well as IL-1beta -induced NF-kappa B activation was not inhibited by 50 µM rottlerin (Fig. 3, panel A). These results demonstrate that the rottlerin effect is specific only for the TNFalpha signaling pathway, and indicate that the NF-kappa B activation in response to neutrophil stimulation with TNFalpha is mediated by PI-PLC and PKCdelta dependent pathways.


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Fig. 3.   Rottlerin inhibition of NF-kappa B activation in human neutrophils is specific for TNFalpha -signaling pathway. A, autoradiograph of NF-kappa B DNA binding in TNFalpha -, LPS-, and IL-1beta -stimulated neutrophils preincubated with rottlerin. Neutrophils were preincubated 15 min with rottlerin (50 µM), and then stimulated 30 min with TNFalpha (10 ng/ml), LPS (10 µg/ml), or IL-1beta (10 ng/ml). NF-kappa B DNA binding was measured by EMSA. B, Western blot analysis of cytoplasmic extracts prepared from neutrophils preincubated 15 min with rottlerin (50 µM), and stimulated 30 min with TNFalpha (10 ng/ml), LPS (10 µg/ml), or IL-1beta (10 ng/ml). Expression of Ikappa Balpha was visualized using Ikappa Balpha -specific polyclonal antibody (top panel). To confirm equal protein loading and transfer to nitrocellulose, the membrane was stripped and reprobed with actin antibody (lower panel). Both panels are representative of three experiments.

Rottlerin Directly Inhibits PKCdelta Kinase Activity in Vitro-- Rottlerin was originally reported to inhibit PKCdelta by competing for ATP binding (17). To confirm that the same rottlerin concentrations inhibiting NF-kappa B activation in TNFalpha -stimulated neutrophils can also inhibit activity of PKCdelta , the PKCdelta was immunoprecipitated from whole cell lysates using PKCdelta specific polyclonal antibody, and PKCdelta kinase activity was measured using histone H1 as a substrate. For comparative purposes, immunoprecipitation using irrelevant glucocorticoid receptor (GR) antibody was performed as a control. As seen in Fig. 4A, while no histone phosphorylation was detected in lysates prepared from TNFalpha -stimulated neutrophils and immunoprecipitated with GR antibody (lane 1), immunoprecipitation with PKCdelta antibody resulted in strong phosphorylation of histone H1 (lanes 2-5), demonstrating that the immunoprecipitation of PKCdelta was specific.


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Fig. 4.   Rottlerin concentrations inhibiting NF-kappa B activation in TNFalpha -stimulated neutrophils inhibit in vitro kinase activity of PKCdelta . A, autoradiograph of PKCdelta activity in TNFalpha -stimulated neutrophils preincubated with rottlerin in vivo. PKCdelta activity was measured as incorporation of 32P into histone H1 in immunoprecipitates prepared from TNFalpha -stimulated (10 ng/ml, 15 min) neutrophils preincubated 15 min with varying concentrations of rottlerin (top panel). The lower panel shows Coomassie staining of the corresponding histone bands. Lane 1 represents immunoprecipitation from TNFalpha -stimulated neutrophils using irrelevant GR antibody. B, autoradiograph of PKCdelta activity immunoprecipitated from TNFalpha -stimulated (10 ng/ml, 15 min) neutrophils, measured with varying concentrations of rottlerin added to the kinase reaction in vitro, before the addition of ATP. The top panel shows 32P incorporation into histone H1, while the lower panel represents Coomassie staining of the corresponding histone bands. The results are representative of three experiments.

To determine whether rottlerin inhibits activity of PKCdelta directly, or whether it inhibits events upstream of PKCdelta , we performed two types of experiments. In the first set of experiments, PKCdelta was immunoprecipitated from TNFalpha -stimulated neutrophils and incubated with rottlerin in vitro (Fig. 4B). Rottlerin inhibited PKCdelta activity in a dose-dependent manner, the IC50 being about 10 µM, which is consistent with the rottlerin inhibition of NF-kappa B activation demonstrated above (Fig. 2B).

In the second set of experiments, neutrophils were preincubated with varying concentrations of rottlerin in vivo, prior to stimulation with TNFalpha , and PKCdelta was immunoprecipitated from corresponding cell lysates and assayed for histone phosphorylation (panel A). Since it is very likely that the extensive washing of the immunoprecipitates efficiently removes rottlerin from PKCdelta , this experiment differentiates between the direct and indirect effect of rottlerin on PKCdelta activity. If rottlerin targets protein(s) (for example, another protein kinase(s)) upstream of PKCdelta , then neutrophil preincubation with rottlerin in vivo would result in a reduced activity of the immunoprecipitated PKCdelta . However, as seen in Fig. 4A, neutrophil preincubation with varying concentrations of rottlerin in vivo did not significantly inhibit histone phosphorylation by the immunoprecipitated PKCdelta . These results demonstrate that the effect of rottlerin on PKCdelta is direct, and further suggest that the activity of PKCdelta is required for NF-kappa B activation in response to neutrophil stimulation with TNFalpha .

Inhibition of NF-kappa B Activation by Rottlerin Is Mediated through Increased Stability of Ikappa Balpha -- To determine whether PKCdelta activates NF-kappa B through regulating cellular pools of the Ikappa Balpha inhibitor, neutrophils were stimulated with TNFalpha (15 min, 10 ng/ml) in the presence of varying concentrations of rottlerin, and cytoplasmic extracts were analyzed by Western blotting using Ikappa Balpha specific polyclonal antibody (Fig. 5A). Consistent with a previous report (11), neutrophil stimulation with TNFalpha substantially reduced the cytosolic pool of Ikappa Balpha (lane 2). Importantly, neutrophil pretreatment with rottlerin inhibited, in a dose-dependent manner, the TNFalpha -induced depletion of cytosolic Ikappa Balpha (Fig. 5A). The lower lane shows reprobing the membrane with control anti-actin antibody, demonstrating equal protein loading and transfer to nitrocellulose.


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Fig. 5.   Inhibition of PKCdelta suppresses degradation of Ikappa Balpha . A, Western blot analysis of cytoplasmic extracts prepared from TNFalpha -stimulated (10 ng/ml, 15 min) neutrophils pretreated 15 min with varying concentrations of rottlerin. Expression of Ikappa Balpha was visualized using Ikappa Balpha -specific polyclonal antibody (top panel). To confirm equal protein loading, the membrane was stripped and reprobed with actin antibody (lower panel). B, Western analysis of cytoplasmic Ikappa Balpha expression in TNFalpha -stimulated (10 ng/ml, 30 min) neutrophils preincubated with rottlerin (50 µM, 15 min) with and without prior pretreatment with cycloheximide (100 µg/ml, 10 min). C, autoradiograph of EMSA of NF-kappa B DNA binding activity measured in nuclear extracts from TNFalpha -stimulated neutrophils incubated with and without rottlerin and cycloheximide as in panel B. Each panel is representative of three experiments.

To determine whether the increased cytoplasmic pools of Ikappa Balpha by rottlerin resulted from new protein synthesis or increased protein stability, neutrophils were pretreated with cycloheximide (100 µg/ml, 10 min) prior to incubation with rottlerin (50 µM, 15 min) and stimulation with TNFalpha (10 ng/ml, 30 min). As seen in Fig. 5, panels B and C, no new protein synthesis was required for the rottlerin up-regulation of Ikappa Balpha and inhibition of NF-kappa B DNA binding, respectively. These results indicate that PKCdelta is involved in the activation of NF-kappa B in response to neutrophil stimulation with TNFalpha by activating pathway(s) leading to degradation of Ikappa Balpha .

To confirm that the PKCdelta involvement in NF-kappa B activation is specific for TNFalpha signaling (Fig. 3), neutrophils were preincubated with rottlerin (50 µM, 15 min) and stimulated 30 min with TNFalpha , LPS, or IL-1beta , and cytoplasmic extracts were analyzed for Ikappa Balpha expression (Fig. 3B). Consistent with NF-kappa B DNA binding (Fig. 3A), the rottlerin effect was specific only for TNFalpha , since the LPS- and IL-1beta -induced Ikappa Balpha degradation were not inhibited (Fig. 3B).

NF-kappa B Activation in the Neutrophil Is Not Regulated by Nuclear Translocation of NF-kappa B Subunits but Correlates with Nuclear Pools of Ikappa Balpha -- In most mammalian cells, activation of NF-kappa B has been shown to be controlled at the level of nuclear translocation of NF-kappa B proteins through their tightly regulated association with Ikappa Balpha anchored in the cytoplasm (21-23). Therefore, we sought to determine whether the inhibition of PKCdelta -dependent activation of NF-kappa B in response to neutrophil stimulation with TNFalpha is mediated by cytoplasmic retention of p50 and p65 NF-kappa B subunits. Neutrophils were stimulated with TNFalpha with and without pretreatment with rottlerin, and the cytoplasmic and nuclear fractions were analyzed by Western blotting using p50- and p65-specific antibodies. Surprisingly, both the cytoplasmic and the nuclear levels of p50 and p65 NF-kappa B subunits were not significantly affected by neutrophil stimulation with TNFalpha or pretreatment with rottlerin (Fig. 6). Moreover, both NF-kappa B proteins were present in significant amounts in the nucleus even under conditions when the NF-kappa B DNA binding is inhibited: in the control unstimulated neutrophils and in the TNFalpha -stimulated neutrophils pretreated with rottlerin (Fig. 5). To exclude any possible cross-contamination of the cytoplasmic and nuclear fractions, the membrane was stripped and reprobed with antibodies specific for cytoplasmic (LDH) and nuclear (small ubiquitin-related modifier, SUMO-1) proteins. That LDH was detected only in the cytoplasmic fraction, and SUMO-1 in the nuclear fraction (Fig. 6), demonstrates that both fractions were reasonably exempt from cross-contamination.


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Fig. 6.   NF-kappa B activation in the neutrophil is not regulated by nuclear translocation of NF-kappa B subunits but correlates with the amount of Ikappa Balpha in the nucleus, as well as in the cytoplasm. Neutrophils were stimulated with TNFalpha (10 ng/ml, 15 min) with and without preincubation with rottlerin (50 µM, 15 min). Cytoplasmic and nuclear extracts were fractionated and analyzed by Western blotting using polyclonal antibodies specific for p65 and p50 NF-kappa B, and Ikappa Balpha . To evaluate the presence of cytoplasmic proteins in nuclear fractions, the membrane was stripped and reprobed with LDH antibody. Nuclear contamination in cytoplasmic fraction was assessed using SUMO-1 specific antibody. The blot is representative of three experiments.

The presence of p50 and p65 NF-kappa B proteins in the nuclear fraction in the absence of NF-kappa B DNA binding prompted us to investigate the subcellular distribution of Ikappa Balpha . As we hypothesized, significant amounts of Ikappa Balpha were present in the nuclear fraction of resting, unstimulated neutrophils, and neutrophil pretreatment with rottlerin inhibited TNFalpha -induced degradation of Ikappa Balpha in both the nuclear and cytoplasmic compartment (Fig. 6). These results demonstrate that the extent of NF-kappa B activation in the neutrophil is not regulated by the nuclear translocation of its subunits but correlates with nuclear pools of Ikappa Balpha .

Ikappa Balpha Nuclear Localization in Neutrophils Versus Monocytes-- Since the presence of Ikappa Balpha in the nuclear fraction of resting unstimulated neutrophils challenges the current model of NF-kappa B activation, it was important to determine whether this is specific for neutrophils, or whether Ikappa Balpha localizes in the nucleus of other inflammatory cells as well. To address this point, neutrophils and peripheral blood monocytes, other type of inflammatory cells, were analyzed for Ikappa Balpha expression in the nuclear and cytoplasmic fractions of control unstimulated cells, cells stimulated with TNFalpha , and cells pretreated with proteasome inhibitor MG-132 before TNFalpha stimulation. As shown in Fig. 7A, in contrast to neutrophils, in monocytes Ikappa Balpha is predominantly cytoplasmic, with the Ikappa Balpha amount in the nucleus being barely detectable. To evaluate the nucleocytoplasmic distribution of Ikappa Balpha in the neutrophils and monocytes more quantitatively, the cytoplasmic and nuclear fractions prepared from resting unstimulated cells were serially diluted and the amount of Ikappa Balpha was determined by Western blots (Fig. 7B). In neutrophils, about 65% of total cellular Ikappa Balpha localizes in the nucleus, while in monocytes it is only about 3% (n = 4, p < 0.001), confirming that the nuclear localization of Ikappa Balpha is specific for the neutrophils.


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Fig. 7.   Nucleocytoplasmic distribution of Ikappa Balpha in neutrophils and monocytes. A, Western analysis of Ikappa Balpha in nuclear (N) and cytoplasmic (C) fractions in neutrophils and monocytes. Lanes 1, unstimulated resting cells; 2, TNFalpha -stimulated (15 min, 10 ng/ml) cells; 3, TNFalpha -stimulated cells preincubated 30 min with MG-132 (200 µM), inhibitor of proteasome. Each lane contains 5 × 105 cells. Blot is representative of three independent experiments. B, quantitative determination of the total amount of Ikappa Balpha in the nucleus and cytoplasm in resting unstimulated neutrophils and monocytes. Nuclear (N) and cytoplasmic (C) fractions were serially diluted and analyzed by Western blotting and densitometry. Only intensities of bands that fell within the linear response of the film were used for calculation. Results are representative of four independent experiments measured in duplicate.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A large body of research has focused on understanding the regulation of NF-kappa B activation in immune and inflammatory cells, but very little is known about regulation of NF-kappa B activity in the neutrophil. In addition, the signaling pathways leading to NF-kappa B activation in response to neutrophil stimulation with TNFalpha have not been delineated. The results of the present study lead to two important conclusions: first, that NF-kappa B activation in human neutrophils stimulated with TNFalpha , but not with IL-1beta or LPS, is mediated through PKCdelta -dependent degradation of Ikappa Balpha ; and second, NF-kappa B activation in human neutrophils is not regulated by nuclear translocation of NF-kappa B subunits as is the case in most mammalian cells (21-23), but correlates with nuclear pools of Ikappa Balpha .

PKCdelta is a novel type of PKC that is activated by DAG but is unresponsive to Ca2+ (20). In addition to PKCdelta , neutrophils have been shown to contain the classical PKCs alpha  and beta , which are both DAG and Ca2+ dependent, and the atypical PKCzeta , which does not require either DAG or Ca2+ for activation, but may be regulated by 3-phosphorylated inositides produced by phosphoinositide 3-kinase (33-35). Among these PKC isoforms, only PKCdelta is specifically inhibited by rottlerin at concentrations lower than 40 µM (15, 17-19). PKCdelta has been shown to be involved in regulation of apoptosis, and inhibition of PKCdelta by rottlerin blocked all parameters of apoptosis in human neutrophils (15, 16). Since the NF-kappa B activation has been recently implicated in the regulation of TNFalpha -induced apoptosis in human neutrophils (14), we have utilized rottlerin to determine whether NF-kappa B activation in response to neutrophil stimulation with TNFalpha involves the PKCdelta -dependent pathway. We demonstrate that the same concentrations of rottlerin specifically inhibiting the in vitro PKCdelta kinase activity from immunoprecipitated neutrophilic lysates (Fig. 4B), also inhibit TNFalpha -induced NF-kappa B activation in the neutrophil (Fig. 2B). Our data indicate that PKCdelta regulates degradation of Ikappa Balpha , since inhibition of PKCdelta resulted in a dose-dependent increased cellular levels of Ikappa Balpha , independent of new protein synthesis (Fig. 5). The rottlerin inhibition of NF-kappa B activation is specific only for TNFalpha induction, since rottlerin has no effect on NF-kappa B DNA binding or Ikappa Balpha degradation in LPS- and IL-1beta -stimulated neutrophils (Fig. 3), suggesting that PKCdelta is involved in Ikappa Balpha degradation only in TNFalpha signaling.

PKCdelta is activated by DAG generated in vivo by PLC (20). In this study we demonstrate that in human neutrophils, PI-PLC, and not PC-PLC is involved in regulation of NF-kappa B, and is likely to be the upstream activator of PKCdelta . With the exception of study by Nick et al. (12) demonstrating that the LPS-induced activation of NF-kappa B involves activation of mitogen-activated protein kinase, the signaling pathways regulating NF-kappa B in human neutrophils have not been delineated. Therefore at present, we do not know the downstream events regulated by PKCdelta and leading to Ikappa Balpha degradation and NF-kappa B activation in TNFalpha -induced neutrophils. One of the critical regulatory steps dictating degradation of Ikappa Balpha are Ikappa B kinase (IKK), which consists of the catalytic subunits alpha  and beta  and the regulatory subunit gamma , and NF-kappa B inducing kinase (NIK) (36). Recent studies demonstrated that in T lymphocytes, NF-kappa B is activated by PKCtheta through stimulation of IKKbeta (37-39). PKCtheta is another member of the novel PKC family that is selectively expressed in skeletal muscle and T lymphocytes, and plays a vital role in T cell stimulation (37). Neutrophils do not possess PKCtheta , and PKCdelta is the only novel PKC isoform expressed in the neutrophil (20). Therefore, it seems likely that PKCdelta stimulates degradation of Ikappa Balpha in the TNFalpha -stimulated neutrophil by activating IKKalpha /beta , and the exact molecular mechanism is currently under investigation. Although PKCdelta has been implicated in the regulation of transcription factors AP1/Jun (40) and Stat 3 (41), to our knowledge, this is the first report demonstrating involvement of PKCdelta in the TNFalpha -induced NF-kappa B activation.

According to current models of NF-kappa B activation, the biological activity of NF-kappa B is controlled through the nuclear translocation of NF-kappa B subunits (21-23). In the present study we demonstrate that in human neutrophils, the nuclear pools of Ikappa Balpha , and not the NF-kappa B subunits, correlate with NF-kappa B DNA binding activity. While the nuclear and cytoplasmic pools of p50 and p65 NF-kappa B proteins were not significantly affected after neutrophil stimulation or inhibition of NF-kappa B DNA binding (Fig. 6), it was the amount of Ikappa Balpha in the nucleus (and cytoplasm) that reflected the state of NF-kappa B activation. Importantly, the nuclear localization of Ikappa Balpha was specific for the neutrophil, since in the peripheral blood monocytes, Ikappa Balpha was mainly cytoplasmic. Nuclear localization of Ikappa Balpha has been recently demonstrated also in cells overexpressing Ikappa Balpha (42, 43) and in stimulated cells (44-46), since activated NF-kappa B can stimulate neotranscription and neosynthesis of Ikappa Balpha (47, 48). This newly synthesized Ikappa Balpha can then enter the nucleus, remove NF-kappa B from gene promoters, and transport it back to the cytoplasm (49-52). In these models, nuclear localization of Ikappa Balpha is induced by stimuli inducing NF-kappa B activity and can be considered as a cellular mechanism terminating the NF-kappa B-dependent transcription. In contrast, nuclear presence of Ikappa Balpha in resting unstimulated neutrophils suggests its protective role against induction of NF-kappa B activation. In this respect it is important to point out that Ikappa Balpha has been detected in the nuclear fraction of unstimulated cells also in peripheral blood T lymphocytes, however, it was resistant to stimulus-induced degradation, and its levels did not correlate with NF-kappa B DNA binding (53). Studies are currently in progress to determine whether the nuclear retention of Ikappa Balpha in resting neutrophils results from its post-translational modification (phosphorylation) and/or whether it is a consequence of its association with other regulatory protein(s).

Neutrophil exposure to TNFalpha resulted in substantial reduction of both cytoplasmic and nuclear Ikappa Balpha , allowing induction of NF-kappa B DNA binding (Figs. 5-7). Whether this signal-dependent reduction of nuclear Ikappa Balpha content results from nuclear-cytoplasmic shuttling of Ikappa Balpha and its degradation in the cytoplasm, or whether the nuclear Ikappa Balpha can be phosphorylated and degraded in situ, remains to be clarified. While further studies are required to delineate the signaling pathways leading to NF-kappa B activation in human neutrophils, this is the first report characterizing the signaling events resulting in NF-kappa B activation in response to neutrophil stimulation with TNFalpha . Our results suggest that the TNFalpha -induced, but not LPS or IL-1beta -induced activation of NF-kappa B in the neutrophil is mediated by PKCdelta -dependent degradation of Ikappa Balpha . We have shown that NF-kappa B activation in the neutrophil is not regulated by nuclear translocation of NF-kappa B p50 and p65 subunits, but correlates with nuclear, as well as cytoplasmic, pools of Ikappa Balpha . These findings are biologically relevant since they suggest that in the neutrophil, the presence of Ikappa Balpha in the nucleus may function as a safeguard against initiation of NF-kappa B-dependent transcription of proinflammatory and anti-apoptotic genes. It will be important to determine whether the exaggerated expression of inflammatory genes seen in the neutrophil-mediated diseases (1, 2) results from de-regulated activation of NF-kappa B caused by the reduced Ikappa Balpha levels in the nucleus. Identification of the key molecular events regulating nuclear retention of Ikappa Balpha in human neutrophils may have major therapeutic implications.

    ACKNOWLEDGEMENTS

We thank Drs. R. Bienkowski and A. Vancura for critical reading of the manuscript.

    FOOTNOTES

* This work was supported in part by a Long Island Jewish Medical Center Faculty Research Award (to I. V.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Long Island Jewish Medical Center, Research Building, B-49, 270-05 76th Ave., New Hyde Park, NY 11040. Tel.: 718-470-3169; Fax: 718-347-3850; E-mail: vancurov@lij.edu.

Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M100234200

    ABBREVIATIONS

The abbreviations used are: TNFalpha , tumor necrosis factor alpha ; DAG, diacylglycerol; EMSA, electrophoretic mobility shift assay; GR, glucocorticoid receptor; Ikappa B, inhibitor kappa B; IKK, Ikappa B kinase; IL, interleukin; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; NF-kappa B, nuclear factor kappa B; NIK, NF-kappa B inducing kinase; PC-PLC phosphatidylcholine-specific phospholipase C, PI-PLC, phosphatidylinositol-specific phospholipase C; PKC, protein kinase C; SUMO-1, small ubiquitin-related modifier.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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