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(Journal of Leukocyte Biology. 2006;80:1512-1521.)
© 2006 by Society for Leukocyte Biology

Regulation of TNF mediated antiapoptotic signaling in human neutrophils: role of {delta}-PKC and ERK1/2

Laurie E. Kilpatrick1, Shuang Sun, DeMauri Mackie, Fred Baik, Haiying Li and Helen M. Korchak

Department of Pediatrics, University of Pennsylvania School of Medicine and the Joseph Stokes Jr. Research Institute, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

1 Correspondence: Immunology Section, Room 1208A Abramson Research Center, Children’s Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, PA 19104, USA. E-mail: kilpatrick{at}email.chop.edu


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ABSTRACT
 
TNF is implicated in the suppression of neutrophil apoptosis during sepsis. Multiple signaling pathways are involved in TNF-mediated antiapoptotic signaling; a role for the MAP kinases (MAPK), ERK1/2, and p38 MAPK has been suggested. Antiapoptotic signaling is mediated principally through TNF receptor-1 (TNFR-1), and the PKC isotype-delta ({delta}-PKC) is a critical regulator of TNFR-1 signaling. {delta}-PKC associates with TNFR-1 in response to TNF and is required for NF{kappa}B activation and inhibition of caspase 3. The role of {delta}-PKC in TNF-mediated activation of MAPK is not known. The purpose of this study was to determine whether the MAPK, ERK1/2, and p38 MAPK are involved in TNF antiapoptotic signaling and whether {delta}-PKC is a key regulator of MAPK activation by TNF. In human neutrophils, TNF activated both p38 MAPK and ERK1/2 principally via TNFR-1. The MEK1/2 inhibitors PD098059 and U0126, but not the p38 MAPK inhibitor SB203580, decreased TNF antiapoptotic signaling as measured by caspase 3 activity. A specific {delta}-PKC antagonist, V1.1{delta}-PKC-Tat peptide, inhibited TNF-mediated ERK1/2 activation, but not p38 MAPK. ERK1/2 inhibition did not alter recruitment of {delta}-PKC to TNFR-1, indicating {delta}-PKC is acting upstream of ERK1/2. In HL-60 cells differentiated to a neutrophilic phenotype, {delta}-PKC depletion by {delta}-PKC siRNA resulted in inhibition of TNF mediated ERK1/2 activation but not p38 MAPK. Thus, ERK1/2, but not p38 MAPK, is an essential component of TNF-mediated antiapoptotic signaling. In human neutrophils, {delta}-PKC is a positive regulator of ERK1/2 activation via TNFR-1 but has no role in p38 MAPK activation.

Key Words: sepsis • MAP kinases • caspase 3 • {delta}-PKC siRNA


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INTRODUCTION
 
Human neutrophils are important in host defense against bacterial infections but can also contribute to the tissue damage of inflammation if not appropriately regulated. Mature neutrophils are endstage cells and have a relatively short life span. Once released into the circulation, neutrophils undergo constitutive apoptosis. During sepsis and other inflammatory diseases, neutrophil apoptosis is attenuated [1 2 3 4 5 6 ]. Enhanced neutrophil survival at the site of inflammation promotes increased bactericidal activity but may also play a role in acute inflammatory damage.

Proinflammatory cytokines such as TNF are important regulators of neutrophil function during the inflammatory response through activation of proinflammatory signaling and suppression of neutrophil apoptosis [7 8 9 10 11 12 13 ]. Neutrophils possess two TNF receptors, a 55-60 kDa (TNFR-1) and a 75-80 kDa (TNFR-2) receptor; proinflammatory and antiapoptotic signaling is regulated principally by TNFR-1 [14 15 16 17 ]. TNF can activate multiple signaling pathways; however, whether TNF signals for cell survival or programmed cell death is dependent on both cell type and cellular environment.

What regulates TNF-mediated antiapoptotic signaling? Possible target signaling pathways include phosphatidylinositol 3-kinase (PI 3-kinase), the transcription factor nuclear factor-{kappa}B (NF{kappa}B), and mitogen-activated protein kinases (MAPK). PI 3-kinase and NF{kappa}B activation are required for TNF-mediated suppression neutrophil apoptosis [7 , 8 ]. The role of the MAPK, extracellular signal-regulated kinase (ERK1/2), and p38 MAPK in this process has yet to be fully elucidated. MAP kinases play an important role in cell proliferation and differentiation, but these kinases also have important regulatory roles in endstage cells such as neutrophils. MAPK have numerous cellular targets, which include membrane and cytoplasmic proteins, as well as transcription factors [18 , 19 ]. Both p38 MAPK and ERK1/2 have important functions in the inflammatory response. These kinases, either independently or through overlapping signaling, have been implicated in the regulation of respiratory burst activity, priming, degranulation, adherence, and cytokine production [20 21 22 23 24 25 26 27 28 ]. Both ERK1/2 and p38 MAPK are thought to be important in controlling neutrophil apoptosis, and ERK1/2 has been shown to be an important regulator of granulocyte macrophage-colony stimulating factor (GM-CSF), lipopolysaccharide (LPS), and interleukin-8 (IL-8) anti-apoptotic signaling [29 , 30 ]. However, whether TNF activates ERK1/2 in human neutrophils is unclear. Several investigators have reported that TNF activates both ERK1/2 and p38 MAPK [22 , 23 , 26 , 31 , 32 ]. Conversely, other studies found that TNF activated p38 MAPK but not ERK1/2 in neutrophils [21 , 28 , 33 , 34 ]. The reason for these discrepancies is not readily apparent but may be related to TNF concentration, cell adherence, or isolation techniques. Furthermore, the molecular mechanisms involved in TNF-mediated activation of MAPK are less well defined than those activating PI 3-kinase and NF{kappa}B.

Previously, we identified {delta}-PKC as a critical regulator of TNF signaling in neutrophils [7 , 8 , 35 ]. {delta}-PKC is required for TNF-mediated inhibition of constitutive apoptosis and activation of NF{kappa}B in neutrophils, but the role of {delta}-PKC in TNF-mediated MAPK activation in the neutrophil is not known. In this study, we determined selective roles for MAP kinases, ERK1/2 but not p38 MAPK, in TNF-mediated antiapoptotic signaling, and established that {delta}-PKC is a key signaling component in TNF-mediated activation of ERK1/2.


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MATERIALS AND METHODS
 
Reagents
Recombinant human TNF{alpha} and mouse monoclonal anti-human TNFR-2 and TNFR-1 blocking antibodies were obtained from R and D Systems (Minneapolis, MN). The mouse monoclonal anti-human CD120a (TNFR-1) was obtained from Cell Sciences (Norwood, MA). Polyclonal rabbit antiphosphoserine and Membrane Blocking Solution were obtained from Zymed Laboratories (San Francisco, CA). Rabbit polyclonal antibodies against Thr202/Tyr204-phosphorylated ERK1/2, ERK1/2, Thr180/Tyr182-phosphorylated p38 MAPK, and p38 MAPK were purchased from Cell Signaling Technology (Beverly, MA). LY294002 was obtained from Calbiochem (San Diego, CA). Polyclonal rabbit anti-human-{delta}-PKC, anti-βII-PKC, {alpha}-PKC and {zeta}-PKC, goat anti-human TNFR-1, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The MAPK inhibitors, PD098059, U0126, SB203580 were obtained from BioMol (Plymouth Meeting, PA). EGTA, goat anti-mouse IgG agarose, Na-orthovanadate, 4-(2-aminoethyl)-benzenesulfonyl fluoride, leupeptin, protease inhibitor cocktail, and phosphatase inhibitor cocktail were obtained from Sigma (St. Louis, MO). SuperSignal ULTRA chemiluminescence substrate, dimethylpimelimidate (DMP), and bicinchoninic acid (BCA) reagents were obtained from Pierce (Rockford, IL).

{delta}-PKC inhibitor peptide synthesis
{delta}-PKC was selectively inhibited using a {delta}V1.1 PKC-Tat peptide antagonist that consists of a peptide derived from the first unique region (V1) of {delta}-PKC (SFNSYELGSL: amino acids 8-17 of {delta}-PKC) coupled to a membrane permeant peptide sequence in the HIV tat gene product (YGRKKRRQRRR: amino acids 47-57 of Tat) according to the method of Mochly-Rosen [36 ]. The {delta}-PKC peptide was cross-linked by an N-terminal Cys-Cys bond to the membrane-permeable Tat peptide. A carrier-carrier dimer of the Tat peptide alone was used as a control. The peptides were synthesized by Mimotopes (Melbourne, Australia) and purified to > 95% by HPLC.

Preparation of human neutrophils
Neutrophils were isolated from heparinized venous blood (10 U/ml) obtained from adult donors following informed consent in accordance with Institutional Review Board protocols at the Children’s Hospital of Philadelphia. Donors were healthy adults over the age of eighteen who were recruited from the Children’s Hospital of Philadelphia community. The study population included both males and females and represented the ethnic population working at Children’s Hospital of Philadelphia. Standard isolation techniques [37 ] were used employing Ficoll-Hypaque centrifugation, followed by dextran sedimentation and hypotonic lysis to remove residual erythrocytes. Cells were suspended in 10 mM HEPES buffer (pH 7.4). Neutrophil purity was greater than 96% as determined by morphology and Giemsa staining, and viability was greater than 98% as determined by trypan blue exclusion.

HL-60 cell culture and {delta}-PKC siRNA
Human promyelocytic HL60 leukemic cells were grown in suspension culture in RPMI 1640 medium supplemented with 2 mM L-glutamine, 1% nonessential amino acids, 1% MEM vitamin solution, 0.1% gentamicin, and 10% heat-inactivated fetal bovine serum (FBS), as described previously [38 ]. HL60 cells were cultured at 37°C in the presence of 1.3% DMSO for 4 days to initiate differentiation to a neutrophil-like phenotype (dHL60 cells) before treatment with siRNA. Cells were resuspended in Opti-MEM I reduced serum medium at a cell concentration of 25 x 106 cells/800 µl. Validated stealth RNAi (Invitrogen) was used to target {delta}-PKC (Target sequence 5'-CCACUACAUCAAGAACCAUGAGUUU-3'). siRNA with equivalent % GC nucleotide content was used as a control. Delivery of stealth siRNA (500 nM) was enhanced by electroporation at 270 V and 500 µFd, followed by culture in RPMI containing 10% heat inactivated FBS for 48 h.

Measurement of ERK1/2 and p38 MAPK phosphorylation
For inhibitor experiments, neutrophils (20 x 106 cells/well) were incubated with blocking antibodies against TNFR1 and TNFR2 or the PI 3-kinase inhibitor LY 294002 (10 µM) for 20 min before the addition of TNF. For experiments examining the role of {delta}-PKC, neutrophils were pretreated with buffer, {delta}V1.1 PKC-Tat peptide (1 µM), or Tat carrier peptide (1 µM) alone for 120 min at room temperature. After incubation with buffer or TNF (50 ng/ml) at 37°C for varying time intervals, the cells were harvested and the cell lysates were prepared. The cells were lysed in lysis buffer containing 10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM Na-orthovanadate, 20 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 1% triton X-100, 5 µg/ml leupeptin, Sigma phosphatase inhibitor cocktail, and Sigma protease inhibitor cocktail. The cell lysates were run on 4-12% SDS-PAGE gels at a protein concentration of 30 µg/lane. MAPK activation was determined by immunoblotting of cell lysates using phospho-specific antibodies for ERK1/2 (Thr202/Tyr204) and p38 MAPK (Thr180/Tyr182). Equal loading of specific MAPKs was confirmed by reprobing membranes using antibodies that recognize both phosphorylated and nonphosphorylated forms of the specific MAPK. MAPK activation was quantitated by densitometry analysis of Western blot analyses using the software SigmaProscan (Jandel/SPSS), and the results are expressed in arbitrary densitometry units (ADU).

Caspase 3 measurements
Caspase 3-like protease activity was measured as described previously [7 ] by monitoring the cleavage of rhodamine 110 bis-(N-CBZ-L-aspartyl-L-gluamyl-L-valyl-L-aspartic acid amine) (Z-DEVD-R110) . Briefly, neutrophils (1.5 x 106/150 µl were pretreated in the absence or presence of the MEK1/2 inhibitors PD098059 (PD, 50 µM) or U0126 (10 µM), or the p38 MAPK inhibitor SB203580 (SB, 10 µM) for 30 min at 37°C before the addition of TNF. Inhibitors were used at concentrations that effectively inhibit MAPK activity in neutrophils [23 , 39 , 40 ]. The neutrophils were cultured for 20 h at 37°C in RPMI-1640 + 10% heat-inactivated FBS. Caspase 3-like protease activity was determined in cell lysates using the EnzChek Caspase-3 Assay kit #2 (Molecular Probes, Eugene, OR). 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).

Immunoprecipitation of TNFR-1
TNFR-1 immunoprecipitation experiments were carried out as described previously [8 , 35 , 41 ]. Briefly, neutrophils (50 x 106 cells/condition), pretreated in the absence or presence of {delta}-PKC or MEK1/2 inhibitors, were incubated with either TNF (50 ng/ml) or buffer for 5 min. The cells were lysed in immunoprecipitation (IP) buffer and vortexed for 20 min at 4°C to solubilize the membrane fraction. The IP buffer consisted of 10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM Na-orthovanadate, 20 µM 4-(2-aminoethyl)- benzenesulfonyl fluoride, 0.2% NP-40, 5 µg/ml leupeptin, Sigma phosphatase inhibitor cocktail, and Sigma protease inhibitor cocktail. Cell lysates were incubated overnight with a mouse monoclonal anti-TNFR-1 cross-linked to anti-mouse IgG agarose with DMP [42 ]. The IgG agarose pellet was washed, and bound proteins were eluted by incubation with 2x SDS-PAGE sample buffer for 5 min at 95°C. Immunoprecipitated proteins were run on a 4-12% gradient SDS-PAGE and transferred to nitrocellulose membranes. Coimmunoprecipitation of proteins was quantitated by densitometry analysis of Western blot analysis, and the values were expressed in arbitrary densitometry units (ADU).

Statistical analysis
Results are expressed as means ± SE. Data were analyzed by Student’s t-test for two group comparisons or ANOVA for multiple comparisons. The Tukey-Kramer multiple comparisons post-test was used to evaluate the significance between experimental groups. Differences were considered significant when P < 0.05.


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RESULTS
 
TNF-mediated phosphorylation of ERK1/2 and p38 MAPK in neutrophils
We first determined whether TNF activated ERK1/2 and p38 MAPK in isolated neutrophils at a concentration which triggers anti-apoptotic and pro-inflammatory signaling in vitro [7 , 8 , 35 ]. ERK1/2 and p38 MAPK activation was monitored by measurement of phosphorylation of specific tyrosine and threonine residues in the regulatory Thr-Xaa-Tyr site. As shown in Fig. 1 , TNF triggered phosphorylation of both ERK2 and p38 MAPK. Neither ERK2 nor p38 MAPK was activated in isolated neutrophils in the absence of stimuli. Both ERK1 and ERK2 were present in neutrophils and the addition of TNF triggered significant tyrosine phosphorylation of ERK2 that remained elevated over the 60 min incubation period (Fig. 1A) . Phosphorylation was observed as early as 1 min, but maximal phosphorylation was not reached until 5 min incubation time (data not shown). ERK1 was also tyrosine phosphorylated in response to TNF, but phosphorylation was over ninefold less than ERK2 (ERK2 = 10609 ± 934 ADU (arbitrary densitometry units) vs. ERK1 = 1146 ± 116 ADU, n=4). In human neutrophils, enhanced tyrosine phosphorylation of ERK2 as compared with ERK1 in response to TNF has been previously reported [22 , 43 ].


Figure 1
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  		Figure 1. Time course of TNF-mediated activation of ERK1/2 and p38 MAPK in neutrophils. (A) Time course of TNF-mediated ERK activation was determined in cell lysates by immunoblotting with antibodies specific to phosphorylated and activated ERK2 (p-ERK2). ERK2 activation in response to TNF (50 ng/ml) was determined over a 60-min incubation period. Equal protein loading was determined by Western blot analysis for total ERK1/2. Representative Western blot analysis of 4 separate neutrophil preparations. (B) Time course of TNF-mediated activation of p38 MAPK was determined in cell lysates by immunoblotting with antibodies specific to phosphorylated and activated p38 MAPK (phos-p38 MAPK) as described in Materials and Methods. Equal protein loading was determined by Western blot analysis for total p38 MAPK. Representative Western blot analysis of 4 neutrophil preparations.

TNF-mediated phosphorylation of p38 MAPK followed similar kinetics as ERK2 activation, maximal phosphorylation was attained by 5 min, and phosphorylation remained elevated over the 60-min incubation period (Fig. 1B) . Thus, at a concentration of 50 ng/ml, TNF triggered phosphorylation of both ERK1/2 and p38 MAPK in suspended human neutrophils.

Role of MAPK in TNF mediated inhibition of neutrophil apoptosis
Activation of caspases is one of the earliest markers of apoptosis. Caspase 3 is activated during neutrophil constitutive apoptosis, and this activation occurs upstream of DNA cleavage in the apoptotic pathway. Caspase 3 is the final downstream effector caspase mediating apoptosis and is thought to be the first step in the execution phase of apoptosis and the irreversible commitment of the cell to apoptosis. We and others have demonstrated that caspase 3 plays a critical role in spontaneous neutrophil apoptosis [7 , 44 , 45 ]. Previous studies demonstrated that TNF decreased both neutrophil DNA fragmentation (TUNEL) and caspase 3 activity in isolated neutrophils [7 ]. As shown in Fig. 2 , there is little caspase 3 activity in freshly isolated neutrophils. The culture of neutrophils for 20 h resulted in a sixfold increase in Caspase 3-like activity as compared with freshly isolated neutrophils. The addition of TNF to neutrophil cultures for 20 h decreased Caspase 3 activity to 45 ± 4% of neutrophils cultured in buffer alone. Pretreatment with the MEK1/2 inhibitors PD098059 or U0126 prior to the addition of TNF abolished the inhibitory effect of TNF on Caspase 3 activity. In contrast, preincubation with the p38 MAPK inhibitor SB203580 had no significant effect on TNF-mediated suppression of Caspase 3 activity. Thus, ERK1/2 but not p38 MAPK activation is required for TNF antiapoptotic signaling.


Figure 2
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  		Figure 2. TNF-mediated suppression of Caspase 3 activity: Role for ERK1/2 Caspase 3 activity was determined fluorometrically by monitoring cleavage of a fluorescent-labeled substrate peptide in cell lysates prepared from neutrophils cultured for 0 and 20 h. Neutrophils were cultured in the absence or presence of TNF (50 ng/ml). The MAPK inhibitors, PD098059 (PD, 50 µM), U0126 (10 µM), and SB203580 (SB, 10 µM) were added 1 h before the addition of TNF. Results are means ± SE from 5 separate neutrophil preparations, each performed in triplicate. *, P < 0.001: Buffer (0 time) vs. Buffer (20 h); **, P < 0.01: buffer (20 h) vs. TNF, buffer (20 h) vs. TNF+SB; ***, P < 0.01 TNF + PD098059 vs. TNF and TNF+U0126 vs. TNF.

Phosphorylation of ERK1/2 and p38 MAPK by TNF is mediated principally through TNFR-1
Neutrophils possess two different TNF receptors, TNFR-1 and TNFR-2, and both receptors are involved in TNF signaling. We next examined the contributions of TNFR-1 and TNFR-2 in phosphorylation of ERK1/2 and p38 MAPK. As shown in Fig. 3A , phosphorylation of ERK2 is mediated principally by TNFR-1. Similar to ERK2, phosphorylation of ERK1 was also mediated by TNFR1 (TNF+TNFR1 blocking Ab = 42 ± 5% of TNF alone, P < 0.01 and TNF+ TNFR2 blocking Ab = 115 ± 17% of TNF alone, P = NS, n = 4). TNF-mediated phosphorylation of p38 MAPK was also dependent on TNFR-1 initiated signaling (Fig. 3B) . However, in contrast to ERK2 and ERK1, both TNFR-1 and TNFR-2 are involved in p38 MAPK signaling.


Figure 3
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  		Figure 3. Activation of ERK2 and p38 MAPK by TNF is mediated principally through TNFR-1. Neutrophils were incubated in the absence or presence of blocking antibodies to TNFR1 (R1, 10 µg/ml) or TNFR2 (R2, 10 µg/ml) for 30 min at 37°C. Neutrophils were then incubated with buffer or TNF (50 ng/ml) for 5 min. (A) TNF-mediated ERK2 activation was determined in cell lysates by immunoblotting with antibodies specific to phosphorylated and activated ERK2 (p-ERK2). Equal protein loading was determined by Western blot analysis for total ERK1/2. (Representative Western blot analysis from four separate experiments.) Densitometry analysis of TNF-mediated ERK2 activation in the absence or presence of blocking antibodies to TNFR-1(R1) and TNFR-2 (R2). Values are expressed as means ± SE (n = 4 separate neutrophil preparations) and are expressed in arbitrary densitometry units. *Statistical significance P < 0.01 buffer vs. TNF and buffer vs. TNF+R2, **, P < 0.01 TNF vs. TNF + R1 and TNF+R2 vs. TNF + R1. (B) TNF-mediated p38 MAPK activation was determined in cell lysates by immunoblotting with antibodies specific to phosphorylated and activated p38 MAPK (phos-p38MAPK). Equal protein loading was determined by Western blot analysis for total p38 MAPK. (Representative Western blot analysis from four separate experiments.) Densitometry analysis of TNF-mediated p38 MAPK activation in the absence or presence of blocking antibodies to TNFR-1(R1) and TNFR-2 (R2). Values are expressed as means ± SE (n=4 separate neutrophil preparations) and are expressed in arbitrary densitometry units. *, P < 0.01 buffer vs. TNF; **, P < 0.01 buffer vs. TNF+R2 and TNF vs. TNF + R2; ***, P < 0.01 buffer vs. TNF+R1, TNF vs. TNF+R1 and TNF + R1 vs. TNF + R2.

TNF-mediated activation of ERK1/2 and p38 MAPK: Role of {delta}-PKC
TNF triggers phosphorylation of TNFR-1 on multiple serine and threonine residues [35 , 46 47 48 ]. Both ERK1/2 and {delta}-PKC are regulators of TNF-mediated phosphorylation of TNFR-1 [35 , 47 , 49 ]. Similar to ERK1/2, {delta}-PKC is also required for TNF-mediated suppression of caspase 3 activity in neutrophils, suggesting ERK1/2 and {delta}-PKC may be components of the same signaling pathway [7 ]. A possible site of ERK1/2 and {delta}-PKC interaction is at the level of the TNFR-1 signaling complex where both {delta}-PKC and ERK1/2 are recruited to TNFR-1 in response to TNF [8 , 35 , 47 , 50 ]. To determine whether ERK1/2 and {delta}-PKC are components of a linear or parallel signaling pathways, we next determined the role of ERK1/2 in TNF-mediated recruitment of {delta}-PKC to TNFR-1. As shown in Fig. 4 , TNF triggers the recruitment of {delta}-PKC to the TNFR-1 complex within 5 min of incubation with TNF. Pretreatment of neutrophils with the MEK1/2 inhibitor PD098059 (50 µM) had no significant effect on {delta}-PKC recruitment to TNFR-1, indicating that ERK1/2 does not regulate TNF-mediated activation of {delta}-PKC.


Figure 4
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  		Figure 4. Recruitment of {delta}-PKC to TNFR-1 Signaling Complex is not mediated by ERK1/2. (A) Recruitment of {delta}-PKC to TNFR-1: Role of ERK1/2. Neutrophils were pretreated with buffer or the MEK1/2 inhibitor PD098059 (PD, 50 µM) before the addition of TNF (50 ng/ml). TNFR-1 was immunoprecipitated after 5 min incubation with buffer or TNF, as described in Materials and Methods. Co-immunoprecipitation of {delta}-PKC with TNFR-1 was determined by Western blot analysis. Densitometry analysis of TNF mediated recruitment of {delta}-PKC to TNFR-1 in the absence or presence of the PD098059. Values are expressed as means ± SE (n=3 separate neutrophil preparations) and are expressed in arbitrary densitometry units. *Statistical significance P < 0.01 buffer vs. TNF, **, P < 0.01 buffer vs. TNF + PD. (B) Recruitment of {delta}-PKC to TNFR-1 and serine phosphorylation of TNFR-1. Neutrophils were incubated in the absence or presence of the specific {delta}-PKC inhibitor {delta}V1.1 PKC-Tat peptide (1 µM) or the Tat peptide (1 µM) before the addition of TNF. Cells were incubated with buffer or TNF (50 ng/ml) for 5 min before immunoprecipitation. Coimmunoprecipitation of {delta}-PKC with TNFR-1 was determined by Western blot analysis. Serine phosphorylation of TNFR-1 was determined using an antiphosphoserine antibody. Shown are representative Western blots from 5 separate experiments.

We next examined the role of {delta}-PKC in TNF-mediated activation of ERK1/2 and p38 MAPK. Pretreatment of cells with {delta}V1.1 PKC-Tat peptide, a {delta}-PKC antagonist, which inhibits translocation and activation of {delta}-PKC [36 ], significantly inhibited TNF-mediated recruitment of {delta}-PKC to TNFR-1 and associated serine phosphorylation of TNFR-1 (Fig. 4B) . Conversely, pretreatment with the TAT carrier peptide alone had no significant effect on TNF-mediated recruitment of {delta}-PKC to TNFR-1 signaling complex or on serine phosphorylation. Furthermore, incubation with either {delta}V1.1 PKC-Tat peptide or the Tat carrier had no significant effect on TNFR-1 immunoprecipitation as demonstrated by equal receptor concentrations under the different experimental conditions. Thus, recruitment of {delta}-PKC to TNFR-1 is associated with TNF-mediated serine phosphorylation of TNFR-1.

TNF triggered phosphorylation of ERK2 was significantly depressed when neutrophils were pretreated with the {delta}V1.1 PKC-Tat peptide as compared with neutrophils treated with TNF alone or TNF + Tat carrier (Fig. 5A ). Pretreatment with the TAT carrier peptide alone had no significant effect on TNF-mediated phosphorylation of ERK2. Similar to ERK2, TNF-mediated ERK1 phosphorylation was also significantly depressed after pretreatment with {delta}V1.1 PKC-Tat peptide (TNF+{delta}V1.1 PKC-Tat peptide = 53 ± 9% of TNF alone and 56 ± 9% of TNF+Tat peptide, P < 0.01 and TNF+Tat peptide = 94 ± 7% of TNF alone, P = NS, n = 4). Conversely, {delta}V1.1 PKC-Tat pretreatment had no significant effect on TNF-mediated activation of p38 MAPK (Fig. 5B) . PI 3-kinase is also involved in TNF-mediated suppression of caspase 3 activity. To ascertain whether PI 3-kinase had a role in TNF-mediated MAPK signaling, we examined the effect of the PI 3-kinase inhibitor on ERK2 and p38 MAPK phosphorylation. As shown in Fig. 5 , LY 294002 had no significant effect on either ERK2 or p38 MAPK, indicating TNF-mediated activation of either ERK2 or p38 MAPK is PI 3-kinase independent. Thus, {delta}-PKC is a positive regulator of ERK1/2 activation but has no regulatory role in p38 MAPK activation.


Figure 5
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  		Figure 5. TNF-mediated activation of ERK1/2 and p38 MAPK: Role of {delta}-PKC and PI 3-kinase TNF-mediated phosphorylation of ERK2 and p38 MAPK was determined in neutrophils incubated in the absence or presence of the specific {delta}-PKC inhibitor {delta}V1.1 PKC-Tat peptide (1 µM), Tat carrier peptide (1 µM) or LY 294002 (10 µM) before the addition of TNF. Neutrophils were then incubated with buffer or TNF for 5 min. ERK2 and p38 MAPK activation was determined by Western blot analysis using phosphospecific ERK1/2 and p38 MAPK antibodies as described in Fig. 1 . (A) ERK2 activation: *, P < 0.01 buffer vs. TNF; **, P < 0.01 buffer vs. TNF+Tat; ***, buffer vs. TNF+{delta}PKC-Tat, TNF+Tat vs. TNF+{delta}PKC-Tat, TNF vs. TNF+{delta}PKC-Tat; and #, P < 0.01 buffer vs. TNF+LY (n = 4). (B) p38 MAPK Activation: *, P < 0.01, vs. buffer (n=4).

Depletion of {delta}-PKC by siRNA in dHL-60 cells: effect on TNF-mediated activation of MAPK
To further confirm the role of {delta}-PKC in TNF-mediated activation of ERK1/2, HL60 cells differentiated to a neutrophilic phenotype were depleted of {delta}-PKC. dHL-60 cells also contain the PKC isotypes {alpha}, βII, and {zeta} [38 , 51 ]. Pretreatment with Stealth {delta}-PKC siRNA selectively depleted {delta}-PKC, but not {alpha}-, βII-, or {zeta}-PKC (Fig. 6A ). Similar to neutrophils, TNF activates both ERK1/2 and p38 MAPK in dHL60 cells (Fig. 6B and 6C) . In dHL60 cells transfected with the GC control siRNA, the addition of TNF (50 ng/ml) resulted in a 10-fold increase in ERK2 phosphorylation, similar to what is observed in human neutrophils (Fig. 1) . As shown in Fig. 6B , TNF-mediated ERK2 phosphorylation in dHL60 cells depleted of {delta}-PKC was significantly decreased as compared with GC controls (47% of GC control, P < 0.01). TNF-mediated ERK1 phosphorylation was also significantly depressed in {delta}-PKC depleted cells as compared with those treated with GC control siRNA ({delta}-PKC siRNA+ TNF = 49 ± 6% of GC siRNA+ TNF, P < 0.001, n = 4). TNF also triggered phosphorylation of p38 MAPK in dHL60 cells. The level of p38 MAPK phosphorylation in response to TNF was comparable in dHL60 cells transfected with either GC control siRNA or {delta}-PKC siRNA (P = NS, Fig. 6C ). These results provide further evidence of the regulatory role of {delta}-PKC in TNF-mediated ERK1/2 activation but not in p38 MAPK activation.


Figure 6
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  		Figure 6. Effect of {delta}-PKC depletion by siRNA on ERK1/2 and p38 MAPK activation in differentiated HL-60 cells. (A) Selective depletion of {delta}-PKC by stealth {delta}-PKC siRNA in differentiated HL60 (dHL60) cells. dHL60 cells were depleted of {delta}-PKC by treatment with {delta}-PKC stealth siRNA ({delta}-PKC siRNA), as described in Materials and Methods. dHL60 cells treated with siRNA with equivalent % GC nucleotide content (GC-control) as the stealth {delta}-PKC siRNA, were used as controls. Levels of specific PKC isotypes were determined in cell lysates by immunoblotting with isotype-specific antibodies to {alpha}-PKC, β-PKC, {delta}-PKC, and {zeta}-PKC. (Representative Western blots from 4 separate experiments). (B) Effect of {delta}-PKC depletion on ERK2 activation. dHL-60 cells were incubated in the presence or absence of TNF (50 ng/ml) after transfection with {delta}-PKC stealth siRNA ({delta}-PKC siRNA) or with siRNA containing equivalent % GC nucleotide content (GC-control). TNF-mediated ERK2 activation was determined in dHL60 cell lysates, as described in Fig. 1 . Representative Western blots from 4 separate experiments. Densitometry analysis of TNF-mediated ERK2 activation in dHL60 cells transfected with GC control siRNA or {delta}-PKC siRNA. Values are expressed as means ± SE (n=4) and are expressed in arbitrary densitometry units (ADU). *Statistical significance P < 0.01, GC Cont vs. GC+ TNF; **, P < 0.01, {delta}-PKC Cont vs. {delta}-PKC+TNF and GC+TNF vs. {delta}-PKC + TNF. (C) Effect of {delta}-PKC depletion on p38 MAPK activation. TNF-mediated p38 MAPK activation was determined in dHL60 cell lysates as described in Fig. 1 . Representative Western blots from 4 separate experiments. Densitometry analysis of TNF-mediated p38 MAPK activation in dHL60 cells transfected with GC control siRNA or {delta}-PKC siRNA. Values are expressed as means ± SE (n=4) and are expressed in arbitrary densitometry units. *, P < 0.01, GC Cont vs. GC+ TNF and {delta}-PKC Cont vs. {delta}-PKC+TNF.


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DISCUSSION
 
The results of the present study demonstrate that TNF, at a concentration that triggers proinflammatory and antiapoptotic signaling, activates both ERK1/2 and p38 MAPK in human neutrophils. ERK1/2, but not p38 MAPK, is an important component of TNF antiapoptotic signaling and is required for TNF-mediated suppression of caspase 3 activity. Furthermore, {delta}-PKC, an important regulator of TNF-antiapoptotic signaling, is required for TNF-triggered activation of ERK1/2, but not p38 MAPK, indicating differential regulation of these MAP kinases by TNF.

Similar to TNF, other proinflammatory cytokines and mediators suppress neutrophil constitutive apoptosis in an ERK1/2-dependent but p38 MAPK-independent manner. A regulatory role for ERK1/2 has been indicated for antiapoptotic signaling by LPS, IL-8, GM-CSF and LTB4 [29 , 30 , 52 ]. p38 MAPK was not a component of the antiapoptotic signaling pathway for LPS, LTB4, IL-8, or GM-CSF. These results support the hypothesis that ERK1/2 activation is a component of a common signaling pathway required for suppression of neutrophil apoptosis by proinflammatory mediators [29 ]. Inhibition of constitutive apoptosis by proinflammatory mediators is dependent on NF{kappa}B, PI 3-kinase and ERK1/2 and the relative involvement of a particular anti-apoptotic pathway appears to be mediator dependent. The inhibitory site(s) of constitutive apoptosis has yet to be identified but may involve IL-8, activation of antiapoptotic members of the bcl-2 family and IAP (inhibitors of apoptosis), as well as inhibition of proapoptotic members of the bcl-2 family [1 , 10 , 53 54 55 ].

In contrast to other proinflammatory mediators, TNF is a unique cytokine whose signaling pathways are linked to both proapoptotic and antiapoptotic responses (for reviews, see [56 , 57 ]. Both pro- and antiapoptotic signaling pathways are regulated principally by TNFR-1, and the neutrophil response to TNF is dependent on both the intracellular and external cellular environment. TNF-mediated activation of both ERK1/2 and p38 MAPK is also mediated principally via TNFR-1. These findings are consistent with previous studies that demonstrated in other cell types, a requirement for TNFR-1 in p38 MAPK and ERK1/2 activation [58 59 60 ]. Activation of ERK1/2 does not appear to require TNFR-2 (Fig. 3 and Ref. [61 ] ). However, a role for TNFR-2 in sustained activation of ERK1/2 has been suggested [62 ] and our studies, performed at an earlier time point, do not rule out the involvement of TNFR2 at later times. In contrast to ERK1/2, activation of p38 MAPK was dependent on TNFR-2 signaling (Fig. 3B) . In mouse macrophages, deletion of either TNF receptor also inhibited p38 MAPK activation [62 ]. Thus, while both ERK1/2 and p38 MAPK are mediated principally via TNFR-1, they are discrete pathways that are under differential regulation by TNF. Furthermore, only ERK1/2 is required for antiapoptotic signaling. These MAPKs are activated by distinct signaling cascades or modules and the precise activation pathway by TNF has yet to be fully elucidated.

PI 3-kinase activation is associated with cell survival and PI 3-kinase activation is required for TNF-mediated suppression of apoptosis and caspase 3 activity [7 , 10 , 53 ]. The present study demonstrates that TNF-mediated activation of ERK1/2 and p38 MAPK are PI 3-kinase independent. TNF is an incomplete secretagogue and requires cell adherence to matrix proteins and engagement of β-integrins to trigger superoxide anion generation, degranulation, and activation of the kinases c-Jun N-terminal kinase and PI 3-kinase [12 , 41 , 63 ]. Our studies demonstrate that TNF activates ERK1/2 and p38 MAPK in suspended neutrophils (Fig. 1) , and activation was not altered by pretreatment with the PI 3-kinase inhibitor prior to the addition of TNF (Fig. 5) . Thus, TNF-mediated activation of ERK1/2 and p38 MAPK does not require cooperative signaling between β-integrins and TNF signaling.

Both ERK1/2 and {delta}-PKC are required components of TNF antiapoptotic signaling (Fig. 2 and Ref. [7 ]). {delta}-PKC is a critical regulator of TNF signaling mediated through TNFR-1 and is required for TNF-mediated activation of NF{kappa}B and the suppression of neutrophil apoptosis [7 , 8 , 35 ]. Using a highly specific {delta}-PKC inhibitor in primary neutrophils and siRNA in dHL60 cells, we established a selective role for {delta}-PKC in TNF-mediated activation of ERK1/2 but not p38 MAPK. A regulatory role for {delta}-PKC has been implicated in growth factors and GPCR (G protein-coupled receptor) agonist-mediated ERK1/2 activation. However, this appears to be agonist and cell type specific [64 65 66 67 ]. Moreover, {delta}-PKC can activate ERK1/2 by several different mechanisms and can act at several different points in the ras/raf/MEK/ERK pathway [68 ]. Our studies demonstrate that while {delta}-PKC regulates ERK1/2 activation, ERK1/2 is not involved in TNF-mediated recruitment of {delta}-PKC to TNFR-1, indicating that signaling is not bidirectional and {delta}-PKC is acting upstream of ERK1/2 activation. However, these studies do not rule out the possibility that {delta}-PKC may act at multiple sites in the ERK1/2 signal transduction pathway.

At what point {delta}-PKC regulates TNF-mediated ERK1/2 activation remains to be defined but may be at the level of TNFR-1 receptor complex. {delta}-PKC is an important regulator of TNFR-1 signaling, it is activated by TNF, associates with TNFR-1 signaling complex, and is required for TNF-mediated serine phosphorylation of TNFR-1 [7 , 8 , 35 ]. {delta}-PKC is a positive regulator of TNF mediated NF{kappa}B activation through control of assembly of the TNFR1-TRADD-RIP-TRAF2 complex [7 , 8 ]. Less is known about the signaling complex required for TNF-mediated activation of ERK1/2. {delta}-PKC can regulate TRAF2 and RIP recruitment to TNFR-1 [8 ]. RIP is an important component of TNFR-1 signaling complex for activation of either p38 MAPK or ERK1/2 [69 , 70 ]. Moreover, ERK activation requires RIP kinase activity, but p38 MAPK activation is independent of kinase activity [70 , 71 ]. Thus, a possible regulatory role for {delta}-PKC may be through regulation of RIP kinase activity. Alternatively, both {delta}-PKC and ERK1/2 have been implicated in TNF-mediated phosphorylation, and receptor phosphorylation is an important mechanism for regulation of receptor function. TNF triggers phosphorylation of TNFR-1 on both serine and threonine residues and serine phosphorylation of TNFR-1 is important in regulatory control of TNF-mediated activation of NF{kappa}B and inhibition of apoptosis [7 , 8 , 35 , 46 47 48 49 50 ]. {delta}-PKC may mediate phosphorylation of TNFR-1 through control of ERK1/2 activation. Which serine residues on TNFR-1 are under {delta}-PKC control have yet to be identified. It is not known whether these serine residues are the same phosphorylated by ERK1/2 or are linked through hierarchal phosphorylation of TNFR-1 to ERK1/2 activation.

In summary, {delta}-PKC is an important regulator of TNFR-1-based signaling in neutrophils and is a positive regulator of ERK1/2 activation, but not p38 MAPK. These results indicate differential regulation of TNFR-1 signaling by {delta}-PKC, perhaps through serine phosphorylation of the receptor. The ability of {delta}-PKC to regulate ERK1/2 activation may be key to TNF antiapoptotic signaling. An understanding of TNF signaling in the neutrophil is highly relevant for control of tissue damage at the site of inflammatory loci. A better understanding of these signaling events will enable design of a targeted strategy to control tissue damage in inflammatory diseases.


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ACKNOWLEDGEMENTS
 
This work was supported by Grants GM 64552 (L. E. K.) and AI 24840 (H. M. K) from the National Institutes of Health, Bethesda, MD.

Received April 20, 2006; revised June 20, 2006; accepted June 29, 2006.


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