INTRODUCTION

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HUMAN NEUTROPHILS are important in host defense against bacterial infection but may also contribute to the tissue damage of inflammation. Neutrophils ingest and kill invading microorganisms through the release of toxic oxygen radicals and proteases. Mature human neutrophils have a relatively short life span in the circulation and undergo spontaneous or constitutive apoptosis within 24 h. This apoptotic process is thought to be a protective mechanism that minimizes the risk of host tissue damage caused by the release of neutrophil-derived toxic mediators. Neutrophils undergoing apoptosis have decreased oxygen radical production, degranulation, and phagocytosis in response to stimuli (24, 57).

During inflammatory diseases such as sepsis, systemic inflammatory response syndrome (SIRS), and adult respiratory distress syndrome (ARDS), neutrophil apoptosis is attenuated (14, 22, 23, 34, 37). The increased survival of neutrophils may contribute to the pathophysiology of these inflammatory diseases through excessive release of toxic mediators, resulting in host tissue damage and organ dysfunction. Tumor necrosis factor (TNF)- and other proinflammatory cytokines have been implicated as endogenous mediators responsible for the modulation of neutrophil apoptosis during inflammation (10, 12, 14, 18, 22, 23, 27, 31, 34, 37, 39, 42, 54).

TNF- is a unique proinflammatory cytokine whose signaling pathways are linked to both antiapoptotic and proapoptotic responses in neutrophils (3, 10, 12, 23, 39, 42, 54). Neutrophils possess two TNF- receptors, a 55-60 kDa (p60TNFR) and a 75-80 kDa (p80TNFR), and both antiapoptotic and proapoptotic signaling pathways are regulated principally by the p60TNFR (4, 33, 49). This seemingly paradoxical situation suggests that other factors, either intracellular or external, determine whether TNF- will activate signaling pathways for cell survival or programmed cell death.

TNF- is an incomplete secretagogue in neutrophils and requires input from both ligated integrins and TNF- receptors to trigger superoxide anion generation, degranulation, and activation of enzymes such as phosphatidylinositol (PI) 3-kinase (26, 28, 41). The adherence of neutrophils to matrix proteins such as fibronectin provides signaling from -integrins, a requirement for neutrophil responsiveness to TNF- (40, 41, 53). Neutrophils adherent to matrix proteins, rather than neutrophils in suspension, are relevant to an inflammatory focus. Because adherent neutrophils respond differently to TNF- than those in suspension, it is possible that input from integrin-activated signaling may have a critical role in regulating TNF--mediated antiapoptotic signaling. Indeed, different extracellular matrix proteins have disparate effects on neutrophil spontaneous apoptosis and may have an important role in regulating TNF- antiapoptotic and proapoptotic signaling (47, 56).

Our recent studies demonstrate that both protein kinase C- (PKC-) and PI 3-kinase associate with the p60TNFR in response to TNF-, an association that requires engagement of -integrins (26, 28). PI 3-kinase has been implicated in antiapoptotic signaling triggered by proinflammatory cytokines (12, 27). PI 3-kinase activation has been linked to the activation of PKC- in several cell systems (5, 50, 51). PKC- is a phosphatidylserine (PS)/diglyceride (DG)-dependent, calcium-independent PKC isotype. In neutrophils, PKC- has a selective role in the regulation of TNF--mediated signaling through serine phosphorylation of the p60TNFR (26). PKC- is an important signaling component in TNF--mediated proapoptotic signaling (13, 17). Whether PKC- also has an antiapoptotic role in TNF--mediated signaling remains to be determined. The goal of this study was to examine the role of PKC- and PI 3-kinase in TNF--mediated antiapoptotic signaling in neutrophils adherent to the physiologically relevant matrix protein fibronectin.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents. Recombinant human TNF- was obtained from R&D Systems (Minneapolis, MN). Rottlerin, Go-6976, and KN-62 were obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). The PI 3-kinase inhibitor LY-294002 was purchased from Calbiochem (San Diego, CA). Z-VAD-FMK [Z-Val-Ala-Asp(OMe)-FMK] was obtained from Enzyme Systems Products (Livermore, CA). Neutrophil DNA fragmentation was quantitated using an In Situ Cell Death Detection Kit (Boehringer Mannheim, Indianapolis, IN). Caspase-3 activity was determined using the EnzChek caspase-3 assay kit no. 2 (Molecular Probes, Eugene, OR). Human plasma fibronectin was purchased from Life Technologies (Gaithersburg, MD), and SuperSignal ULTRA chemiluminescence substrate was from Pierce (Rockford, IL). Polyclonal rabbit anti-p65 NFB, NFB gel shift oligonucleotides, TransCruz Gel supershift p65 NFB reagent, and peroxidase-conjugated goat anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal goat antilactate dehydrogenase was obtained from Fitzgerald Industries International (Concord, MA), and polyclonal rabbit antihistone H3 was from Upstate Biotechnology (Lake Placid, NY). BSA, EGTA, leupeptin, protease inhibitor cocktail, and phosphatase inhibitor cocktail were obtained from Sigma (St. Louis, MO).

Preparation of human neutrophils and culture. Neutrophils were isolated from heparinized venous blood (10 U/ml) obtained from healthy adult volunteers. Standard isolation techniques (8) were used employing Ficoll-Hypaque centrifugation, followed by dextran sedimentation and hypotonic lysis to remove residual erythrocytes. Cells were suspended in a HEPES buffer (pH 7.3) having the following composition (in mM): 150 Na⁺, 5 K⁺, 1.29 Ca²⁺, 1.2 Mg²⁺, 155 Cl, and 10 HEPES. For cell culture experiments, cells were resuspended in RPMI 1640 supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1% minimal essential medium vitamin solution, 0.1% gentamycin, and 10% heat-inactivated fetal bovine serum. Neutrophils were cultured for 20 h at 37°C in a 5% CO₂ atmosphere in fibronectin-coated 96-well plates at a concentration of 1.5 × 10⁶ cells/200 µl. Fibronectin-coated wells were prepared according to the method of Nathan (41) using a concentration of 3.4 µg/well.

TUNEL assay for DNA fragmentation. Neutrophil DNA fragmentation was quantitated by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling method (TUNEL). Neutrophils (1.5 × 10⁶) were cultured for 20 h in RPMI 1640 plus 10% heat-inactivated FBS in fibronectin-coated 96-well plates. Cells were harvested, washed once in PBS, and fixed in 2% paraformaldehyde/PBS (pH 7.4). The cells were permeabilized in 0.1% Triton X-100/0.1% sodium citrate, incubated with TUNEL reaction mixture containing Tdt and fluorescein labeled dUTP, washed, and resuspended in PBS. Mean channel fluorescence of labeled cells was determined by flow cytometry using an Epics Elite Flow Cytometer equipped with an argon laser. The data are expressed as a percentage of apoptotic neutrophils, and the data were accumulated and stored as two-parameter histogram files. All photomultiplier tube voltages were adjusted to achieve the same mean channel fluorescence values as determined from previous calculations. For each sample, 10,000-20,000 events were accumulated.

Caspase-3 assay. Neutrophils were cultured for 20 h and harvested as described above. The activities of caspase-3-like proteases were determined using a caspase-3 assay kit. Caspase-3-like protease activity was determined by monitoring the cleavage of rhodamine-110 bis-(N-CBZ-L-aspartyl-L-gluamyl-L-valyl-L-aspartic acid amine) (Z-DEVD-R110). Fluorescence was measured in a Fluorocount Microplate Reader (Packard Instruments) at an excitation wavelength of 485 nm and emission of 530 nm. Background fluorescence was determined measuring substrate cleavage in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. Results are expressed as arbitrary fluorescence units (AFU).

Preparation of nuclear fractions. Neutrophils were incubated at 37°C in fibronectin-coated 12-well plates at a concentration of 20 × 10⁶ cells/well. Appropriate inhibitors were added 20 min before the addition of either TNF- or buffer. Samples were then incubated with either TNF- (25 ng/ml) or buffer for 10 min and placed on ice. Nuclear extracts were prepared by a modification of the method of Dignam et al. (11), using KCl instead of NaCl. Briefly, samples were resuspended in hypotonic buffer containing 10 mM HEPES (pH 7.8), 1.5 mM MgCl₂, 10 mM KCl, 1.5 mM EDTA, 0.5 mM DTT, 5 µg/ml leupeptin, Sigma phosphatase inhibitor cocktail, and Sigma protease inhibitor cocktail and were placed on ice for 30 min. Cells were disrupted by Dounce homogenization, and the cellular extract was centrifuged. The nuclear pellet was resusupended in high-salt buffer consisting of 20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl₂, 1.2 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 5 µg/ml leupeptin, Sigma phosphatase inhibitor cocktail, and Sigma protease inhibitor cocktail and were incubated on ice for 20 min. The extract was centrifuged, and aliquots of the supernatant fractions (nuclear extract) were frozen at 70°C. Samples for Western blot analysis were prepared by mixing an aliquot of the nuclear extracts with 2× sample buffer and heating for 15 min at 65°C. Protein concentration was determined using the Noninterfering Protein Assay Kit (GenoTechnology, St. Louis, MO). Purity of nuclear fractions was routinely determined by probing fractions for cytoplasmic (lactate dehydrogenase) and nuclear (histone) markers.

Western blotting. Nuclear extracts were run on a 4-12% gradient SDS-PAGE, transferred to a nitrocellulose membrane, and blocked for 1 h at room temperature with Tris-buffered saline, pH 7.5, containing 0.1% Tween-20 and 1% BSA/3% casein as described previously (26). The membranes were incubated with a rabbit polyclonal anti-p65 NFB antibody, washed, and incubated with peroxidase-conjugated goat anti-rabbit IgG. Immunoreactive bands were visualized using Pierce SuperSignal ULTRA chemiluminescence substrate. Translocation of the p65 NFB subunit to the nucleus was quantitated by densitometry analysis of Western blots by Scan Pro, and the values were expressed in arbitrary densitometry units (ADU).

Electrophoretic mobility shift assay. Reactions for electrophoretic mobility shift assays (EMSAs) were prepared as follows. Nuclear extracts (5 × 10⁵ cell equivalents) were resuspended in a buffer having a final concentration of 10 mM HEPES, pH 7.9, 42 mM NaCl, 120 µM MgCl₂, 20 µM EDTA, 50 µM phenylmethylsulfonyl fluoride, 50 µM DTT, and 12.5% glycerol. A double-stranded oligonucleotide of sequence 5'-AGTTGAGGGGACTTTCCCAGGC-3' was radioactively labeled with [-³²P]ATP (3,000 Ci/mmol). Each reaction mixture contained 10 ng isotope-labeled oligonucleotide probe. For competitions with unlabeled probe, 50-fold greater unlabeled oligonucleotide was added to reactions. For supershift assays, nuclear extracts were pretreated with anti-p65 NFB (C-20). All reagents including 0.5 µg poly(dI-dC) were combined on ice then incubated at room temperature for 2 min. Labeled probe was added last, and reactions were further incubated at room temperature for 10 min. DNA-protein complexes were separated on 7.8% polyacrylamide gels at 4°C with 0.5× TBE as running buffer. Gels were subsequently dried and subjected to autoradiography.

Statistical analysis. Results are expressed as means ± SE. Data were analyzed by Student's t-test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effect of TNF- on spontaneous neutrophil apoptosis: a role for PKC. Freshly isolated human neutrophils were cultured for 20 h on fibronectin-coated wells at 37°C. DNA fragmentation was determined by flow cytometry using the TUNEL method. Under these experimental conditions, neutrophils undergoing spontaneous apoptosis comprised 43 ± 12% of the total neutrophil population (Table 1 and Fig. 1A). The addition of TNF- (25 ng/ml) to neutrophil cultures resulted in a significant attenuation of spontaneous apoptosis compared with neutrophils cultured in medium alone. In the presence of TNF-, DNA fragmentation was significantly reduced by 60% (n = 4, P < 0.01, Table 1 and Fig. 1B).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effect of tumor necrosis factor (TNF)- on neutrophil apoptosis: a role for protein kinase C (PKC)-. DNA fragmentation was determined by flow cytometry using the TUNEL. Neutrophils were cultured for 20 h in fibronectin-coated well plates as described in MATERIALS AND METHODS. Fluorescence histograms are plotted as fluorescence intensity vs. cell number. Percentages indicate apoptotic neutrophils as a percentage of total cell number. Representative histograms are as follows: buffer (A), TNF- (25 ng/ml) (B), TNF- + rottlerin (5 µM) (C), and TNF- + Go-6976 (10 nM) (D).

Effect of TNF- on neutrophil caspase-3 activity: role of PKC. Activation of caspases is one of the earliest markers of apoptosis. Caspase-3 is activated during neutrophil spontaneous apoptosis, and this activation occurs upstream of DNA cleavage in the apoptotic pathway (3, 15, 30, 48). We next determined whether PKC- was acting on TNF--mediated signal transduction at the level of caspase-3 activation. Similar to DNA fragmentation, caspase-3 activity was increased in human neutrophils cultured for 20 h compared with freshly isolated neutrophils (data not shown). As shown in Fig. 2, the culture of neutrophils for 20 h resulted in caspase-3-like activity of 9,802 ± 268 AFU/1.5 × 10⁶ (SE) cells (n = 5). Pretreatment of neutrophils with the peptide caspase inhibitor Z-VAD-FMK (20 µM) for 30 min before the addition of TNF- resulted in a >98 ± 1% inhibition of fluorescence, indicating caspase activity (Fig. 2, P < 0.01). Similar results were also obtained when the more specific caspase-3 inhibitor DEVD-CHO was used (data not shown). The addition of TNF- to neutrophil cultures decreased caspase-3 activity to 50% of neutrophils cultured in buffer alone (P < 0.01, Fig. 2). Pretreatment with rottlerin before the addition of TNF- abolished the inhibitory effect of TNF- on caspase-3 activity (P < 0.01, Fig. 2). In contrast, caspase-3 activity was not significantly different in TNF- + Go-6976-treated samples compared with TNF- treatment alone (P = NS, Fig. 2). Rottlerin is a relatively specific inhibitor of PKC-. At a concentration of 5 µM, rottlerin also inhibits Ca²⁺/calmodulin kinase II but not calcium-dependent PKC isotypes (16, 19). To ascertain whether the effects of rottlerin on caspase-3 activity were Ca²⁺/calmodulin kinase II dependent, the effect of KN-62, a selective inhibitor of Ca²⁺/calmodulin kinase II, was examined. Pretreatment with KN-62 (1 µM) had no significant effect on the antiapoptotic effects of TNF- (93 ± 14% of TNF- alone, P = NS, n = 3), indicating that the inhibitory effect of rottlerin on TNF--mediated antiapoptotic signaling was not Ca²⁺/calmodulin kinase II mediated. Thus, similar to DNA fragmentation, caspase-3 activity was increased in fibronectin-adherent neutrophils cultured for 20 h. The addition of TNF- decreased caspase-3 activity, an effect that was abolished by rottlerin but not Go-6976 or the Ca²⁺/calmodulin kinase II inhibitor KN-62. Therefore, PKC- appears to be selectively involved in TNF--mediated inhibition of neutrophil spontaneous apoptosis and blocks caspase-3 activation.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of TNF- on neutrophil caspase-3 activity: a role for PKC-. Caspase-3 activity was determined fluorometrically by monitoring cleavage of a fluorescent-labeled substrate peptide in cell lysates prepared from fibronectin-adherent neutrophils cultured for 20 h. Neutrophils were cultured in the presence of buffer alone, rottlerin (Rot; 5 µM), Go-6976 (Go; 10 nM), TNF- (25 ng/ml), TNF- + rottlerin, TNF- + Go-6976, or TNF- + Z-VAD-FMK (20 µM). Inhibitors were added 20 min before buffer or TNF-. Results are means ± SE from 5 separate neutrophil preparations and are expressed as arbitrary fluorescence units. *Statistical significance (P < 0.01) compared with buffer alone. **Statistical significance (P < 0.01) compared with TNF- treatment. ***Statistical significance (P < 0.01) compared with either buffer alone or TNF- treatment.

Effect of cycloheximide on TNF--mediated inhibition of caspase-3 activity. To further investigate the mechanism of prolonged neutrophil survival in response to TNF-, the requirement for protein synthesis in fibronectin-adherent neutrophils was next investigated. Inhibition of spontaneous apoptosis by TNF- requires long-term exposure, suggesting that protein synthesis is required for the inhibitory responses. Fibronectin-adherent neutrophils were preincubated with 5 µM cycloheximide before addition of either buffer or TNF-. After a 20-h incubation, cells were harvested, and caspase-3 activity was determined. As shown in Fig. 3, cycloheximide had no significant effect on spontaneous neutrophil apoptosis as determined by caspase-3 activity (P = NS, n = 5). In contrast, cycloheximide pretreatment before TNF- abolished the antiapoptotic effects of TNF- on spontaneous neutrophil apoptosis (P < 0.02, Fig. 3). Thus TNF--mediated attenuation of neutrophil apoptosis in fibronectin-adherent neutrophils requires protein synthesis.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effect of cycloheximide on neutrophil caspase-3 activity. Caspase-3 activity was determined as described in Fig. 2 under the following experimental conditions: buffer alone, cycloheximide (CHX; 5 µM), TNF- (25 ng/ml), or TNF- + CHX. CHX was added 30 min before the addition of either buffer or TNF-. Results are means ± SE from 5 separate neutrophil preparations and are expressed as arbitrary fluorescence units. *Statistical significance (P < 0.01) compared with buffer alone. **Statistical significance (P < 0.02) compared with TNF- treatment.

TNF--mediated activation of NFB: a role for PKC-. TNF- is a potent activator of NFB, a family of transcription factors thought to have a critical role in cell survival through the induction of antiapoptotic genes (1, 6, 7, 9, 32, 44, 55). Neutrophil NFB activation requires the translocation of the p50 and p65 NFB subunits to the nucleus. We next examined whether TNF- activates NFB in fibronectin-adherent neutrophils and whether this activation requires PKC-. As shown in Fig. 4, densitometry analysis of nuclear fractions showed low levels of the p65 NFB subunit present in the nucleus in adherent neutrophils treated with buffer alone. There was no significant increase in translocation of p65 NFB to the nucleus when neutrophils were pretreated with either Go-6976 or rottlerin alone. The addition of TNF- to neutrophils resulted in a threefold increase in p65 NFB translocation (Fig. 4, P < 0.01, n = 5). Pretreatment with the inhibitor Go-6976 had no significant effect on TNF--mediated translocation of p65 NFB. In contrast, pretreatment with rottlerin inhibited TNF--mediated translocation of the p65NFB subunit by 55% (Fig. 4, P < 0.01).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   p65 NFB translocation: a role for PKC-. Neutrophils were incubated in fibronectin-coated 12-well plates (20 × 106 cells/well) under the following conditions: buffer alone, Go-6976 (Go; 10 nM), rottlerin (Rot; 5 µM), TNF- (25 ng/ml), TNF- + Go-6976, or TNF- + rottlerin. Inhibitors were added 20 min before buffer or TNF-. After a 10-min incubation, nuclear extracts were prepared as described in MATERIALS AND METHODS. Nuclear extracts were probed for the presence of p65 NFB by Western blotting. Scanning densitometry of the Western blots was analyzed by ScanPro. Results are expressed as means ± SE (n = 5 separate neutrophil preparations) in arbitrary densitometry units. *Statistical significance (P < 0.01) compared with buffer alone. **Statistical significance (P < 0.05) compared with TNF- treatment. Inset: representative Western blot of the p65 NFB subunit in nuclear extracts.

View larger version (72K): [in this window] [in a new window]   Fig. 5.   NFB DNA-binding activity. NFB DNA-binding activity in nuclear extracts from neutrophils incubated under the following experimental conditions. Neutrophils were treated for 10 min with buffer alone, rottlerin (5 µM), TNF- (25 ng/ml), or TNF- + rottlerin. Rottlerin was added 20 min before addition of buffer or TNF-. Results are representative of 3 separate experiments.

TNF--mediated activation of NFB: a role for PI 3-kinase. In some cell systems, activation of NFB has been linked to PI 3-kinase through a signaling cascade that activates the protein kinase Akt (43). Our recent studies demonstrated that TNF--mediated activation of PI 3-kinase requires twin signals from the p60TNFR and -integrins and that PI 3-kinase is only activated by TNF- in adherent neutrophils (28). We next determined whether PI 3-kinase has a role in TNF--mediated activation of NFB in adherent neutrophils. Pretreatment of neutrophils with the PI 3-kinase inhibitor LY-294002 (10 µM) inhibited TNF--mediated translocation of the p65 NFB subunit to the nucleus by 60% (Fig. 6, P < 0.02, n = 5). LY-294002 itself did not have any effect on p65 NFB translocation. Thus both PKC- and PI 3-kinase are upstream of NFB activation in TNF--mediated signaling in fibronectin-adherent neutrophils.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   TNF--mediated p65 NFB translocation: a role for phosphatidylinositol (PI) 3-kinase. Neutrophils were cultured as described in Fig. 4 under the following experimental conditions: buffer alone, LY-294002 (LY; 10 µM), TNF- (25 ng/ml), or TNF- + LY. Nuclear extracts were probed for p65 NFB by Western blotting. Results are expressed as means ± SE (n = 5 separate neutrophil preparations) and are expressed in arbitrary densitometry units. *Statistical significance (P < 0.01) compared with buffer alone. **Statistical significance (P < 0.02) compared with TNF- treatment. Inset: representative Western blot of the p65 NFB subunit in nuclear extracts.

Effect of PI 3-kinase inhibition on caspase-3 activity. We next determined whether PI 3-kinase had a role in TNF--mediated antiapoptotic signaling in adherent neutrophils. Pretreatment with the PI 3-kinase inhibitor LY-294002 had no significant effect on spontaneous neutrophil apoptosis as determined by caspase-3 activity after a 20-h incubation (Fig. 7, P = NS, n = 6). In contrast, LY-294002 abolished the antiapoptotic effect of TNF- on caspase-3 activity (P < 0.01, Fig. 7). Similar results were also obtained when wortmannin (100 nM) was used to inhibit PI 3-kinase (data not shown). These studies indicate that PI 3-kinase is an essential component of TNF--mediated antiapoptotic signaling.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Effect of PI 3-kinase inhibition on caspase-3 activity. Caspase-3 activity was determined as described in Fig. 2 under the following experimental conditions: buffer alone, LY-294002 (LY; 10 µM), TNF- (25 ng/ml), or TNF- + LY-294002. LY-294002 was added 20 min before the addition of either buffer or TNF-. Results are means ± SE from six separate neutrophil preparations and are expressed as arbitrary fluorescence units. *Statistical significance (P < 0.01) compared with buffer alone. **Statistical significance (P < 0.01) compared with TNF- treatment. AFU, arbitrary fluorescence units.

Effect of inhibition of PKC- and PI 3-kinase on caspase-3 activity. PI 3-kinase and PKC- are required signaling elements for TNF--triggered activation of NFB and inhibition of caspase-3. The effect of combinations of maximal and submaximal concentrations of rottlerin and LY-294002 on TNF--mediated inhibition of caspase-3 activity was examined to determine whether PI 3-kinase and PKC- are components of the same signaling pathway. As shown in Figure 8, both rottlerin and LY-294002 abolish the inhibitory effects of TNF- on caspase-3 activity in a dose-dependent manner. The inhibitory effect of TNF- on caspase-3 activity was completely ablated at 5 µM rottlerin and at 10 µM LY-294002 (P < 0.01, n = 4, Fig. 8). Neither rottlerin, LY-294002, nor the combination of the inhibitors had any significant effect on caspase-3 activity in the absence of TNF- (Figs. 2 and 7, and data not shown). When maximal doses of rottlerin (5 µM) and LY-294002 (10 µM) were used in combination, there was no additive effect, and caspase-3 activity was not significantly different from TNF- plus rottlerin or TNF- plus LY-294002 alone (P = NS, Fig. 8). Furthermore, when submaximal doses of rottlerin and LY-294002 were used in combination, there were no additive effects on caspase activity, providing further evidence that both PI 3-kinase and PKC- are components of the same signaling pathway.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Effect of rottlerin and LY-294002 on TNF--mediated inhibition of caspase-3 activity. Caspase-3 activity was determined as described in Fig. 2 under the following experimental conditions: buffer alone, TNF- (25 ng/ml), TNF- + rottlerin (1-5 µM), TNF- + LY-294002 (2.5-10 µM), or TNF- + rottlerin and LY-294002. Rottlerin and LY-294002 were added 20 min before the addition of either buffer or TNF-. Results are means ± SE from 4 separate neutrophil preparations and are expressed as arbitrary fluorescence units. *Statistical significance (P < 0.01) compared with buffer alone. **Statistical significance (P < 0.05) compared with TNF- treatment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

TNF- is a unique proinflammatory cytokine whose signaling is linked to both antiapoptotic and proapoptotic pathways (3, 10, 12, 23, 39, 42, 54). The cellular response to TNF- is dependent on cytokine levels, length of exposure, and input from other signaling pathways (i.e., engagement of integrins). PKC- is an important signaling component in TNF--mediated proapoptotic signaling (13, 17) and has also been implicated as an important component in neutrophil apoptosis (16, 25, 45). The results of the present study demonstrate that PKC- is also a critical component of TNF--mediated antiapoptotic signaling in adherent neutrophils, a process that may be mediated by NFB and involve PI 3-kinase.

In this study, the role of -PKC in TNF--mediated antiapoptotic signaling was examined in fibronectin-adherent neutrophils. TNF- significantly delayed apoptosis in cultured fibronectin-adherent neutrophils as determined by DNA fragmentation and caspase-3 activation. These results are concordant with previous studies that demonstrated TNF--mediated antiapoptotic signaling in neutrophils adherent to other matrixes (3, 10, 12, 23, 39, 42, 54). Rottlerin, an inhibitor of PKC-, attenuated both TNF--mediated inhibition of DNA fragmentation and caspase-3 activation. The effect was specific for PKC-, since inhibitors of calcium-dependent PKC isotypes or Ca²⁺/calmodulin kinase II had no effect on TNF--mediated antiapoptotic signaling. In adherent neutrophils, PI 3-kinase is also activated by TNF- and PI 3-kinase is an activator of PKC- (28, 51). Inhibition of PI 3-kinase with LY-294002 also attenuated TNF--mediated inhibition of neutrophil constitutive apoptosis. Furthermore, the inhibitory effects of the PKC- and PI 3-kinase inhibitors were not additive when used in combination, indicating that PKC- and PI 3-kinase are components of the same signaling pathway. Thus PKC- and PI 3-kinase are essential elements in TNF--mediated antiapoptotic signaling and block caspase-3 activation.

Long-term incubation is required for TNF--mediated attenuation of neutrophil apoptosis, suggesting that protein synthesis is a required element. External factors such as cytokines may regulate apoptosis through the transient activation of antiapoptotic genes (3). Gene expression can be regulated through modulation of the activity of transcription factors such as NFB (55). Concordant with the activation of transcription factors is the requirement for protein synthesis; our studies demonstrated that preincubation with the protein synthesis inhibitor cycloheximide abolished the inhibitory effects of TNF- on apoptosis in fibronectin-adherent neutrophils.

Modulation of the transcription factor NFB is a possible regulatory site for TNF--mediated attenuation of neutrophil apoptosis. TNF- activates NFB in numerous cell types including neutrophils (38, 55). A role for NFB has been implicated in the pathophysiology of sepsis, ARDS, and SIRS through enhanced gene expression of proinflammatory cytokines, chemokines, adhesion molecules, and inducible nitric oxide synthase and by the attenuation of phagocytic cell apoptosis (1, 6, 7, 9, 32, 44). Enhanced NFB activation has been reported in patients with sepsis, SIRS, and ARDS and is correlated with poor outcome and increased mortality (7, 32, 44). Inhibition of NFB enhances spontaneous neutrophil apoptosis, suggesting that activation of NFB may be an important regulatory site in control of neutrophil apoptosis (42, 55). The putative antiapoptotic genes that are activated by NFB in response to TNF- have yet to be identified; potential candidates include the zinc finger A20, manganese superoxide dismutase, interleukin-8, the prosurvival Bcl-Xi, TRAF-1, and the family of cellular inhibitors of apoptosis (2, 6, 12, 42).

TNF- is a potent activator of NFB in fibronectin-adherent neutrophils. TNF- enhanced both p65 NFB translocation to the nucleus and increased NFB-DNA binding activity. Rottlerin inhibited both TNF--triggered p65 NFB translocation and NFB-DNA binding. The inhibitory effect was selective for PKC- because inhibition of calcium-dependent PKC isotypes had no effect on TNF--triggered NFB activation. Thus PKC- is a required signaling component for TNF--mediated activation of NFB in adherent neutrophils and acts at a site before NFB translocation. A recent study also reported inhibition of TNF--mediated activation of NFB by rottlerin but at a different regulatory site (52). These studies used nonadherent neutrophils and employed rottlerin at a concentration 10-fold greater than employed in this study (50 vs. 5 µM) and at a concentration far above the IC₅₀ for PKC- (3-6 µM) (52). The discrepancy in the two studies may be attributed to different responses to cellular activation by TNF- of adherent neutrophils, compared with those in suspension. Neutrophils in suspension lack input from -integrin signaling pathways and activation of key enzymes such as PI 3-kinase (28). The results presented here demonstrate that in adherent neutrophils, both PKC- and PI 3-kinase are important components for TNF--mediated activation of NFB and the attenuation of neutrophil apoptosis.

Previous studies in other cell types have identified PKC- as having a critical role in TNF--mediated proapoptotic signaling (13, 17). The results of this study indicate that PKC- is also essential for antiapoptotic signaling. Because both antiapoptotic and proapoptotic signaling pathways are mediated principally through the p60TNFR (4, 20, 21), a possible site of interaction is at the level of the receptor. TNF- binding to the p60TNFR leads to the recruitment of TNFR-1-associated death domain protein (TRADD), which acts as a scaffold protein and subsequently recruits TNFR-associated factor-2 (TRAF2), receptor-interacting protein (RIP), and Fas-associated death domain (FADD) to form the p60TNFR signaling complex (20, 21). TRAF2 and RIP regulate antiapoptotic pathways through activation of NFB or p42/p44 extracellular signal-regulated kinase (46, 55). FADD is essential for TNF--induced apoptosis through its association with and activation of caspase-8, thereby initiating apoptosis (4). For PKC- to regulate both anti- and proapoptotic signaling, a likely site of interaction would be before bifurcation of the signaling pathways, i.e., at the level of the p60TNFR or TRADD. In support of this concept, our previous studies have shown that activation of the p60TNFR by TNF- triggers phosphorylation of the receptor on both serine and threonine residues (26). Serine phosphorylation of the receptor is mediated by PKC- (26). Furthermore, both PI 3-kinase and PKC- associate with the p60TNFR signaling complex in response to TNF- in adherent neutrophils (26, 28). PI 3-kinase and its product PI 3,4,5-trisphosphate can activate several novel and atypical PKC isotypes including PKC- (51). Thus it is suggested that in adherent neutrophils, both PKC- and PI 3-kinase may act at the level of the p60TNFR in mediating signaling through regulation of receptor phosphorylation. These studies do not rule out the possibility that -PKC and/or PI 3-kinase may be acting at several discrete locations rather than a single site in the TNF--initiated signaling pathway.

In summary, the results of the present study demonstrate that both PKC- and PI 3-kinase are essential components of the same pathway for TNF--mediated antiapoptotic signaling in adherent neutrophils. Furthermore, both PKC- and PI 3-kinase are required for TNF--mediated activation of the transcription factor NFB, suggesting that PKC- and PI 3-kinase regulate TNF--mediated neutrophil survival through induction of antiapoptotic genes. The requirement for PKC- for both TNF--mediated anti- and proapoptotic signaling suggests that PKC- is acting at a site before bifurcation of these signaling pathways, possibly at the level of the p60TNFR.