NF-κB Activation in Tumor Necrosis Factor α-stimulated Neutrophils Is Mediated by Protein Kinase Cδ

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

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 TNF␣, 1 interleukin (IL)-1␣ and IL-1␤, IL-8, and macrophage inflammatory protein ␣, that may amplify the inflammatory process (3)(4)(5)(6)(7)(8). Expression of many of these proinflammatory proteins is regulated at the level of gene transcription by transcription factor NF-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-B activity in human neutrophils consists of p50/50 homodimers and p50/65 heterodimers, and that their activation in TNF␣-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-B in neutrophils is mediated by p38␣ mitogen-activated protein kinase. However, the signaling pathways leading to NF-B activation in response to neutrophil stimulation with TNF␣ are undefined.
Interestingly, a recent study demonstrated that NF-B also regulates both constitutive and TNF␣-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), PKC␦ (15,16). Therefore, we sought to investigate involvement of PKC␦ in TNF␣-induced activation of NF-B in human neutrophils. PKC␦ is selectively inhibited by rottlerin (15,(17)(18)(19), and as any other novel PKC, it is activated in a Ca 2ϩ -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 PKC␦ blocks activation of NF-B in TNF␣-stimulated human neutrophils by inhibiting degradation of IB␣. The regulation of NF-B activation by PKC␦ is specific only for TNF␣ signaling, since LPS-or IL-1␤-induced activation of NF-B and degradation of IB␣ are not inhibited by rottlerin. In addition, we show that in human neutrophils, but not monocytes, IB␣ localizes in significant amounts in the nucleus of resting unstimulated cells. The NF-B DNA binding in the neutrophil does not correlate with nuclear translocation of NF-B subunits, as is the case in most mammalian cells (21)(22)(23), but rather with the amount of IB␣ in the nucleus, as well as in the cytoplasm.
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 ϫ 10 6 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 Me 2 SO alone, before stimulation with TNF␣. The incubations were terminated by placing cells on ice and rapid centrifugation (1 min, 5,000 ϫ g, 4°C).
The nuclear pellets were washed in 200 l of buffer A containing the protease inhibitors, and re-centrifuged. The pelleted nuclei were resuspended in 50 l of ice-cold nuclear buffer (NE buffer: 20 mM Hepes, pH 7.5, 25% glycerol, 0.8 M KCl, 1 mM MgCl 2 , 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 ϫ g, 15 min, 4°C), and the resulting supernatants (nuclear extracts) were aliquoted and stored at Ϫ80°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 (5Ј-TTGTTACAAGGGGACTTTCCGCTGGGGACTTTCCAGGGAGGC-3Ј) containing two tandemly repeated NF-B-binding sites (underlined). Mutant oligonucleotide used for competition studies was 5Ј-TTGTTA-CAATCTCACTTTCCGCTTCTCACTTTCCAGGGAGGC-3Ј. End labeling was accomplished by treatment with T4 kinase in the presence of [␥-32 P]ATP, and the labeled oligonucleotide was purified on a Sephadex G-25 column, as described elsewhere (25).
Nuclear extracts (containing 4 -6 g of protein in 5-7 l) were incubated (20 min at room temperature) with 5-10 fmol of radiolabeled oligonucleotide (ϳ70,000 cpm) in 20 l 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 g of acetylated bovine serum albumin and 2 g 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 g of specific polyclonal antibody, respectively, and incubated 15 min at room temperature before adding 32 P-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 ϫ 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 Bi-oMax MS) with intensifier screen at Ϫ80°C.
Immunoprecipitation PKC␦ Assay-PKC␦ enzymatic activity was assayed in whole cell lysates immunoprecipitated by PKC␦ specific polyclonal antibody as follows. Neutrophils (5 ϫ 10 6 ) were lysed in 0.3 ml of lysis buffer (50 mM Tris-Cl, pH 8.0, 250 mM NaCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1% Triton X-100, 10% glycerol, 2 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 100 g/ml soybean trypsin inhibitor; 1 mM benzamidine, 2 mM levamisole, 1 mM Na 3 VO 4 , 20 mM glycerophosphate, 10 mM NaF and protease inhibitor mixture from Sigma (P-8340), used at concentration 60 l/5 ϫ 10 6 cells). Soluble proteins were pre-cleared by a 1-h incubation (4°C) with 10 l of Protein A/G Plus-agarose. The precleared supernatants were incubated with 1 g of anti-PKC-␦ or control anti-GR antibody (2 h, 4°C), and immunoprecipitated with 10 l 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 MgCl 2 , 2 mM MnCl 2 , 20 M ATP), and resuspended in 20 l of kinase buffer. Five l of 5 ϫ reaction buffer (1 mg/ml histone H1, 20 M 1,2-dioleoyl-sn-glycerol, and 0.25 mg/ml L-␣-phosphatidyl-L-serine) and 5 Ci of [␥-32 P]ATP were added, and the samples were incubated for 5 min at 30°C. Reactions were stopped by the addition of 8 l of 5 ϫ 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 PKC␦ activity in vitro, rottlerin was added to the PKC␦ immunoprecipitates in concentrations given in the text before the addition of 1 M ATP.
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 IgGhorseradish 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 Ϯ S.E. Statistical significance was evaluated by using ANOVA.

Specific Inhibitors of PI-PLC and PKC␦ Block Activation of NF-B in TNF␣-stimulated Neutrophils-To investigate whether the TNF␣-induced NF-B activation involves PLC-
and PKC␦-dependent pathways, we used inhibitors of phosphatidylcholine (PC)-and phosphatidylinositol (PI)-specific PLC, and PKC␦: D-609 (26), U-73122, and Et-18-OCH3 (27)(28)(29), and rottlerin (15,(17)(18)(19), respectively. Neutrophils were preincubated 15 min with or without the corresponding inhibitor, stimulated 30 min with TNF␣, and the NF-B DNA binding activity was measured in nuclear extracts by EMSA. As seen in Fig. 1A, neutrophil stimulation with TNF␣ 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-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-B heterodimer.
The p50/65 NF-B DNA binding activity was inhibited by U-73122 (5 M) and Et-18-OCH3 (50 M), inhibitors of PI-PLC, and by PKC␦ inhibitor rottlerin (50 M). In contrast, inhibitor of PC-PLC, D-609 at 50 M concentration previously shown to be selectively effective to inhibit PC-PLC activity (26, 31) did not reduce NF-B DNA binding (Fig. 1A). The inhibition of NF-B DNA binding by U-73122 was dose dependent (Fig. 2A). The complete inhibition of p50/65 NF-B was achieved at 5 M U-73122 concentration, and the IC 50 was ϳ2 M. This IC 50 value is consistent with the previously reported IC 50 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 M concentrations, had no effect on NF-B DNA binding (data not shown). Fig. 2B shows a dose response of the PKC␦-specific inhibitor rottlerin on NF-B DNA binding in TNF␣-stimulated neutrophils. The complete inhibition of the p50/65 NF-B heterodimer was achieved by rottlerin concentrations of 50 M, and the IC 50 was ϳ10 M. These values correlate well with the previously reported rottlerin IC 50 for PKC␦ inhibition 3-6 M, whereas the IC 50 values for other PKC isoforms were 40 -100 M (17)(18)(19). In contrast, neutrophil pretreatment with Ro-31-8425 (100 nM), which inhibits the classical isoforms of PKC but not PKC␦ (32), had no inhibitory effect on TNF␣-induced NF-B DNA binding even at concentration 10 times higher than the reported IC 50 (data not shown). Importantly, the inhibitory effect of rottlerin on NF-B DNA binding was specific only for TNF␣ induction, since LPS, as well as IL-1␤-induced NF-B activation was not inhibited by 50 M rottlerin (Fig. 3, panel A). These results demonstrate that the rottlerin effect is specific only for the TNF␣ signaling pathway, and indicate that the NF-B activation in response to neutrophil stimulation with TNF␣ is mediated by PI-PLC and PKC␦ dependent pathways.
Rottlerin Directly Inhibits PKC␦ Kinase Activity in Vitro-Rottlerin was originally reported to inhibit PKC␦ by competing for ATP binding (17). To confirm that the same rottlerin concentrations inhibiting NF-B activation in TNF␣-stimulated neutrophils can also inhibit activity of PKC␦, the PKC␦ was immunoprecipitated from whole cell lysates using PKC␦ specific polyclonal antibody, and PKC␦ 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 TNF␣-stimulated neutrophils and immunoprecipitated with GR antibody (lane 1), immunoprecipitation with PKC␦ antibody resulted in strong phosphorylation of histone H1 (lanes 2-5), demonstrating that the immunoprecipitation of PKC␦ was specific.
To determine whether rottlerin inhibits activity of PKC␦ directly, or whether it inhibits events upstream of PKC␦, we performed two types of experiments. In the first set of experiments, PKC␦ was immunoprecipitated from TNF␣-stimulated neutrophils and incubated with rottlerin in vitro (Fig. 4B). Rottlerin inhibited PKC␦ activity in a dose-dependent manner, the IC 50 being about 10 M, which is consistent with the rottlerin inhibition of NF-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 TNF␣, and PKC␦ 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 PKC␦, this experiment differentiates between the direct and indirect effect of rottlerin on PKC␦ activity. If rottlerin targets protein(s) (for example, another protein kinase(s)) upstream of PKC␦, then neutrophil preincubation with rottlerin in vivo would result in a reduced activity of the immunoprecipitated PKC␦. However, as seen in Fig. 4A, neutrophil preincubation with varying concentrations of rottlerin in vivo did not significantly inhibit histone phosphorylation by the immunoprecipitated PKC␦. These results demonstrate that the effect of rottlerin on PKC␦ is direct, and further suggest that the activity of PKC␦ is required for NF-B activation in response to neutrophil stimulation with TNF␣.
Inhibition of NF-B Activation by Rottlerin Is Mediated through Increased Stability of IB␣-To determine whether PKC␦ activates NF-B through regulating cellular pools of the IB␣ inhibitor, neutrophils were stimulated with TNF␣ (15 min, 10 ng/ml) in the presence of varying concentrations of rottlerin, and cytoplasmic extracts were analyzed by Western blotting using IB␣ specific polyclonal antibody (Fig. 5A). Consistent with a previous report (11), neutrophil stimulation with TNF␣ substantially reduced the cytosolic pool of IB␣ (lane 2). Importantly, neutrophil pretreatment with rottlerin inhibited, in a dose-dependent manner, the TNF␣-induced depletion of cytosolic IB␣ (Fig. 5A). The lower lane shows reprobing the membrane with control anti-actin antibody, demonstrating equal protein loading and transfer to nitrocellulose.
To determine whether the increased cytoplasmic pools of IB␣ by rottlerin resulted from new protein synthesis or in- creased protein stability, neutrophils were pretreated with cycloheximide (100 g/ml, 10 min) prior to incubation with rottlerin (50 M, 15 min) and stimulation with TNF␣ (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 IB␣ and inhibition of NF-B DNA binding, respectively. These results indicate that PKC␦ is involved in the activation of NF-B in response to neutrophil stimulation with TNF␣ by activating pathway(s) leading to degradation of IB␣.

NF-B Activation in the Neutrophil Is Not Regulated by Nuclear Translocation of NF-B Subunits but Correlates with
Nuclear Pools of IB␣-In most mammalian cells, activation of NF-B has been shown to be controlled at the level of nuclear translocation of NF-B proteins through their tightly regulated association with IB␣ anchored in the cytoplasm (21)(22)(23). Therefore, we sought to determine whether the inhibition of PKC␦-dependent activation of NF-B in response to neutrophil stimulation with TNF␣ is mediated by cytoplasmic retention of p50 and p65 NF-B subunits. Neutrophils were stimulated with TNF␣ 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-B subunits were not significantly affected by neutrophil stimulation with TNF␣ or pretreatment with rottlerin (Fig. 6). Moreover, both NF-B proteins were present in significant amounts in the nucleus even under conditions when the NF-B DNA binding is inhibited: in the control unstimulated neutrophils and in the TNF␣-stimulated neutrophils pretreated with rottlerin (Fig. 5). To exclude any possible crosscontamination 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.
The presence of p50 and p65 NF-B proteins in the nuclear fraction in the absence of NF-B DNA binding prompted us to investigate the subcellular distribution of IB␣. As we hypothesized, significant amounts of IB␣ were present in the nuclear fraction of resting, unstimulated neutrophils, and neutrophil pretreatment with rottlerin inhibited TNF␣-induced degradation of IB␣ in both the nuclear and cytoplasmic compartment (Fig. 6). These results demonstrate that the extent of NF-B activation in the neutrophil is not regulated by the nuclear translocation of its subunits but correlates with nuclear pools of IB␣.
IB␣ Nuclear Localization in Neutrophils Versus Monocytes-Since the presence of IB␣ in the nuclear fraction of resting unstimulated neutrophils challenges the current model of NF-B activation, it was important to determine whether this is specific for neutrophils, or whether IB␣ 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 IB␣ expression in the nuclear and cytoplasmic fractions of control unstimulated cells, cells stimulated with TNF␣, and cells pretreated with proteasome inhibitor MG-132 before TNF␣ stimulation. As shown in Fig. 7A, in contrast to neutrophils, in monocytes IB␣ is predominantly cytoplasmic, with the IB␣ amount in the nucleus being barely detectable. To evaluate the nucleocytoplasmic distribution of IB␣ in the neutrophils and monocytes more quantitatively, the cytoplasmic and nuclear fractions prepared from resting unstimulated cells were serially diluted and the amount of IB␣ was determined by Western blots (Fig. 7B). In neutrophils, about 65% of total cellular IB␣ localizes in the nucleus, while in monocytes it is only about 3% (n ϭ 4, p Ͻ 0.001), confirming that the nuclear localization of IB␣ is specific for the neutrophils. DISCUSSION A large body of research has focused on understanding the regulation of NF-B activation in immune and inflammatory cells, but very little is known about regulation of NF-B activity in the neutrophil. In addition, the signaling pathways leading to NF-B activation in response to neutrophil stimulation with TNF␣ have not been delineated. The results of the present study lead to two important conclusions: first, that NF-B activation in human neutrophils stimulated with TNF␣, but not with IL-1␤ or LPS, is mediated through PKC␦-dependent degradation of IB␣; and second, NF-B activation in human neutrophils is not regulated by nuclear translocation of NF-B subunits as is the case in most mammalian cells (21)(22)(23), but correlates with nuclear pools of IB␣.
PKC␦ is a novel type of PKC that is activated by DAG but is unresponsive to Ca 2ϩ (20). In addition to PKC␦, neutrophils have been shown to contain the classical PKCs ␣ and ␤, which are both DAG and Ca 2ϩ dependent, and the atypical PKC, which does not require either DAG or Ca 2ϩ for activation, but may be regulated by 3-phosphorylated inositides produced by phosphoinositide 3-kinase (33)(34)(35). Among these PKC isoforms, only PKC␦ is specifically inhibited by rottlerin at concentrations lower than 40 M (15,(17)(18)(19). PKC␦ has been shown to be involved in regulation of apoptosis, and inhibition of PKC␦ by rottlerin blocked all parameters of apoptosis in human neutrophils (15,16). Since the NF-B activation has been recently implicated in the regulation of TNF␣-induced apoptosis in human neutrophils (14), we have utilized rottlerin to determine whether NF-B activation in response to neutrophil stimulation with TNF␣ involves the PKC␦-dependent pathway. We demonstrate that the same concentrations of rottlerin specifically inhibiting the in vitro PKC␦ kinase activity from immunoprecipitated neutrophilic lysates (Fig. 4B), also inhibit TNF␣-induced NF-B activation in the neutrophil (Fig. 2B). Our data indicate that PKC␦ regulates degradation of IB␣, since inhibition of PKC␦ resulted in a dose-dependent increased cellular levels of IB␣, independent of new protein synthesis (Fig. 5). The rottlerin inhibition of NF-B activation is specific only for TNF␣ induction, since rottlerin has no effect on NF-B DNA binding or IB␣ degradation in LPS-and IL-1␤-stimulated neutrophils (Fig. 3), suggesting that PKC␦ is involved in IB␣ degradation only in TNF␣ signaling.
PKC␦ 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-B, and is likely to be the upstream activator of PKC␦. With the exception of study by Nick et al. (12) demonstrating that the LPS-induced activation of NF-B involves activation of mitogen-activated protein kinase, the signaling pathways regulating NF-B in human neutrophils have not been delineated. Therefore at present, we do not know the downstream events regulated by PKC␦ and leading to IB␣ degradation and NF-B activation in TNF␣-induced neutrophils. One of the critical regulatory steps dictating degradation of IB␣ are IB kinase (IKK), which consists of the catalytic subunits ␣ and ␤ and the regulatory subunit ␥, and NF-B inducing kinase (NIK) (36). Recent studies demonstrated that in T lymphocytes, NF-B is activated by PKC through stimulation of IKK␤ (37)(38)(39). PKC 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 PKC, and PKC␦ is the only novel PKC isoform expressed in the neutrophil (20). Therefore, it seems likely that PKC␦ stimulates degradation of IB␣ in the TNF␣-stimulated neutrophil by activating IKK␣/␤, and the exact molecular mechanism is currently under investigation. Although PKC␦ 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 PKC␦ in the TNF␣-induced NF-B activation.
According to current models of NF-B activation, the biological activity of NF-B is controlled through the nuclear translocation of NF-B subunits (21)(22)(23). In the present study we demonstrate that in human neutrophils, the nuclear pools of IB␣, and not the NF-B subunits, correlate with NF-B DNA binding activity. While the nuclear and cytoplasmic pools of p50 and p65 NF-B proteins were not significantly affected after neutrophil stimulation or inhibition of NF-B DNA binding (Fig. 6), it was the amount of IB␣ in the nucleus (and cytoplasm) that reflected the state of NF-B activation. Importantly, the nuclear localization of IB␣ was specific for the neutrophil, since in the peripheral blood monocytes, IB␣ was mainly cytoplasmic. Nuclear localization of IB␣ has been recently demonstrated also in cells overexpressing IB␣ (42,43) and in stimulated cells (44 -46), since activated NF-B can stimulate neotranscription and neosynthesis of IB␣ (47,48). This newly synthesized IB␣ can then enter the nucleus, remove NF-B from gene promoters, and transport it back to the cytoplasm (49 -52). In these models, nuclear localization of IB␣ is induced by stimuli inducing NF-B activity and can be considered as a cellular mechanism terminating the NF-B-dependent transcription. In contrast, nuclear presence of IB␣ in resting unstimulated neutrophils suggests its protective role against induction of NF-B activation. In this respect it is important to point out that IB␣ 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-B DNA binding (53). Studies are currently in progress to determine whether the nuclear retention of IB␣ 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 TNF␣ resulted in substantial reduction of both cytoplasmic and nuclear IB␣, allowing induction of NF-B DNA binding (Figs. 5-7). Whether this signal-dependent reduction of nuclear IB␣ content results from nuclear-cytoplasmic shuttling of IB␣ and its degradation in the cytoplasm, or whether the nuclear IB␣ can be phosphorylated and degraded in situ, remains to be clarified. While further studies are required to delineate the signaling pathways leading to NF-B activation in human neutrophils, this is the first report characterizing the signaling events resulting in NF-B activation in response to neutrophil stimulation with TNF␣. Our results suggest that the TNF␣-induced, but not LPS or IL-1␤-induced activation of NF-B in the neutrophil is mediated by PKC␦-dependent degradation of IB␣. We have shown that NF-B activation in the neutrophil is not regulated by nuclear translocation of NF-B p50 and p65 subunits, but correlates with nuclear, as well as cytoplasmic, pools of IB␣. These findings are biologically relevant since they suggest that in the neutrophil, the presence of IB␣ in the nucleus may function as a safeguard against initiation of NF-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-medi-ated diseases (1, 2) results from de-regulated activation of NF-B caused by the reduced IB␣ levels in the nucleus. Identification of the key molecular events regulating nuclear retention of IB␣ in human neutrophils may have major therapeutic implications.