Departments of 1Physiology and 2Surgery, and Burn and Shock Trauma Institute, Stritch School of Medicine, Loyola University Medical Center, Maywood, Illinois
Submitted 30 June 2004 ; accepted in final form 18 December 2004
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ABSTRACT |
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polymorphonuclear neutrophils; nuclear factor-B; Bcl-xl; Bad; inhibitory apoptosis protein; burn injury
While spontaneous neutrophil apoptosis has been shown to be delayed in the presence of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF, tumor necrosis factor- (TNF-
) is known to both promote and delay PMN apoptosis (2, 35, 36). The release of such mediators is known to occur in burn injury and can cause delay in neutrophil apoptosis leading to exacerbation of inflammation after burns (10). Our previous study (15) has shown that a delay in neutrophil apoptosis after burn injury is accompanied by suppression of mitochondrial apoptotic mechanisms. To date, no studies have provided any significant information as to neutrophil signaling mechanisms and their effects on anti-/proapoptotic targets that might be relevant to the delay in apoptosis with burns.
Phosphatidylinositol-3 kinase (PI3-kinase)/protein kinase B (PKB) has been shown to be a key signal transducer for survival factors such as growth factors, cytokines, and integrins in various cell types (14, 38). PKB is a major mediator of survival signals downstream of PI3-kinase (19). Its activity is positively regulated by phosphorylation on residues Thr308 and Ser473 downstream of PI3-kinase. PKB phosphorylates Bad at the Ser136 residue that is critical for sequestration to 14-3-3 (a cytosolic molecular chaperone) (41). After apoptotic stimuli, Bad is released from 14-3-3 and subsequently dimerizes with the anti-apoptotic protein Bcl-xl. Because Bcl-xl resides constitutively bound to Bax, Bad displaces and releases Bax from Bcl-xl. Thereafter, Bax translocates to the mitochondria and promotes release of cytochrome c, formation of the apoptosome, and activation of the caspase cascade (22).
G-CSF is known to prolong neutrophil survival by inhibiting activation of the caspase cascade. The effect of G-CSF could be abolished by cycloheximide, suggesting that protein synthesis is required for the cytokine's antiapoptotic effect. Previous studies (23, 37) have also shown that Bcl-xl and inhibitory apoptosis protein (IAP) family proteins are direct targets for transcriptional regulation by NF-B. Bcl-xl promotes cell survival via formation of regulatory mitochondrial inner membrane channels that inhibit the release of cytochrome c from mitochondria to cytosol (27, 40). The IAP family includes cellular IAP1 (cIAP1), cIAP2, X-linked IAP (XIAP), and survivin. TNF upregulates cIAP2 through activation of NF-
B in Jurkat cells (5). The expression of survivin is increased by stimulation with G-CSF and granulocyte-macrophage-CSF (3, 12).
In this study, we evaluated the role of PI3-kinase in the delay of apoptosis of neutrophils from burn-injured rats by assessing PKB phosphorylation. Specifically, we ascertained the downstream events of PI3-kinase/PKB activation in neutrophils during early vs. later culture periods.
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MATERIALS AND METHODS |
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Neutrophil isolation and culturing.
Blood (1012 ml) was collected through cardiac puncture into heparinized syringes. Neutrophils were isolated from heparinized blood via Ficoll-Paque (Pharmacia, Peapack, NJ) gradient centrifugation. The erythrocyte/granulocyte pellet was diluted 1:1 with normal saline. Erythrocytes were sedimented by 3% dextran (Sigma, St. Louis, MO) in 0.9% saline and incubated for 1 h. Supernatants were collected and centrifuged for 10 min at 4°C. Remaining red blood cells were lysed in distilled water. The freshly isolated neutrophils were resuspended in modified RPMI 1640 (Cellgro Mediatech, Herndon, VA) containing 10% heat-inactivated fetal calf serum (Cellgro Mediatech), 100 U/ml penicillin, 100 µg/ml streptomycin, and 300 µg/ml glutamine (GIBCO-BRL, Grand Island, NY). The neutrophil preparation routinely contained 95% neutrophils as identified by the Giemsa stain and were found to be
98% viable by the Trypan blue exclusion technique. Five milliliters of cell suspension were put into each well of 6-well plates (Fisher Scientific, Pittsburgh, PA), which were then incubated at 37°C with or without 100 nM wortmannin (Calbiochem), 10 µM LY-294002 (Calbiochem), and cycloheximide (CHX) (Sigma) (1, 5, and 10 µg/ml) for the indicated periods in a humidified incubator containing 95% atmosphere and 5% CO2. To evaluate the effect of injury-related agonists modulating apoptosis in vivo, neutrophils were stimulated with 100 nM N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (Sigma) before being assessed by phosphorylation of PKB and Bad and for determining translocation of NF-
B in vitro.
Annexin V analysis of neutrophil apoptosis. Neutrophil apoptosis was measured by flow cytometry with the annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis assay kit (Pharmingen/BD Biosciences, San Diego, CA). The experiment was performed by following the manufacturer's instructions with minor changes. Briefly, after isolation or incubation, neutrophils were washed twice with ice-cold PBS and then resuspended in binding buffer. Neutrophils were analyzed by flow cytometry within 1 h of annexin V-PI labeling. Viable neutrophils were defined as negative for annexin V-FITC and PI staining; apoptotic neutrophils were defined as positive for annexin V-FITC but negative for PI staining; necrotic cells were defined as positive for both annexin V and PI staining. Cell apoptosis was expressed as a percentage of apoptotic neutophils in relation to the counted neutrophils.
Confocal laser scanning microscopy. Immunohistochemical staining for nuclei and Rel A was performed. After isolation or incubation, 106 neutrophils were washed once in ice-cold PBS and were fixed with 2% (wt/vol) paraformyldehyde in PBS for 15 min at room temperature, washed twice in PBS, and permeabilized in buffer containing 1% Triton X-100 (wt/vol) and 1% (wt/vol) bovine serum albumin (Sigma) in PBS. Neutrophils were incubated with antibody against Rel A (Cell Signaling, Beverly, MA) or resuspended in buffer without antibody (as negative control of staining). After incubation, neutrophils were washed twice and resuspended in secondary antibody (Alexa 488-conjugated goat-anti-rabbit IgG; Molecular Probes, Eugene, OR) at a final concentration of 5.0 µg/ml. Incubation with the secondary antibody was for 30 min. After three washes in PBS, neutrophils were RNase treated and counterstained with PI to label DNA for nuclear localization. Cytospins were prepared on glass slides, air dried, and mounted in a mounting medium (Baxter Healthcare, Deerfield, IL). The slides were analyzed with a confocal laser-scanning microscope.
RNA extraction, RT-PCR, and real-time PCR. Total RNA was isolated from neutrophils with RNeasy Mini Kit according to the manufacturer's instructions (Qiagen, Valencia, CA). Neutrophils were washed twice with ice-cold PBS and then lysed in Buffer RLT. The lysates were pipetted directly onto a Qiashredder spin column placed in a 2-ml collection tube and centrifuged for 2 min at maximum speed. After centrifugation, 600 µl of 70% ethanol were added to the homogenized lysates and mixed well by pipetting. Further DNA removal was performed with Qiagen RNase-free DNase set. RNase-free water (30 µl) was pipetted onto the RNeasy silica membrane, and the resulting RNA was in the elution.
To generate cDNA, 2 µg of total RNA were used for each reaction. The reaction mixtures (20 µl) contained RNA, oligo(dT) primer, dNTP mixture, and Omniscript reverse transciptase (Qiagen). The reaction mixtures were incubated for 60 min at 37°C. One-tenth of the synthesized cDNA was then amplified. PCR reaction mixtures (25 µl) contained cDNA, dNTP mixture, MgCl2 (1.5 mM), Taq DNA polymerase (2.5 U/µl), and forward and reverse primers. The following six primers were used: rat cIAP1 (forward: 5'-ACATTTCCCCAGCTGCCCATTC-3', reverse: 5'-CTCCTGCTCCGTCTGCTCCTCT-3'); rat cIAP2 (forward: 5'-CCAGCCTGCCCTCA AACCCTCT-3', reverse: 5'-GGGTCATCTCCGGGTTCCCAAC-3'); rat XIAP (forward: 5'-CGCGAGCGGGGTTTCTCTACAC-3', reverse: 5'-ACCAGGCACGGT CACAGGGTTC-3'); rat survivin (forward: 5'-CAACCTGGACCTGAGTGACAT-3', reverse: 5'-CCACCCATAGATCCTGTCAGA-3'); and rat GAPDH (forward: 5'-CCATCA CCATCTTCCAGGAG-3', reverse: 5'-CCTGCTCACCACCTTCTTG-3'). The samples were denatured at 95°C for 15 min and amplified for 30 cycles (RGAPDH) or 32 cycles (cIAP1, cIAP2, XIAP, and survivin), with the last cycle extended at 72°C for 10 min. Samples were resolved on a 2% agarose gel and visualized with ethidium bromide.
Real-time PCR analysis of IAPs was performed on a GeneAmp 5700 sequence detection system (Applied Biosytems). Platinum QPCR Supermix-UDG (Life Technologies, Grand Island, NY), 12.5 µM Rox reference dye (Life Technologies), SYBR Green DNA binding dye diluted 1:2,000, forward and reverse primers (2.5 µM in 10 mM Tris), RNase- and DNase-free H2O, and appropriate sample cDNA were added to each sample. Negative controls included samples without reverse transcriptase or RNA or cDNA. Controls, standards, and samples were run in triplicate (20 µl volume) in a 96-well optical reaction plate and capped with optical caps (Applied Biosystems). The real-time PCR thermal cycler profile was run as follows: one cycle at 50°C for 2 min, one cycle at 95°C for 10 min, 40 cycles of denaturing at 95°C for 15 s, and annealing and elongation at 60°C for 1 min, followed by a dissociation protocol run to test the melting temperature of the product. The data were analyzed using GeneAmp 5700 SDS software (Applied Biosystems). In each experiment, GAPDH PCR products were used an endogenous reference to calculate the relative amount of IAP mRNA.
Western blot analysis.
The whole cell lysates were obtained as follows. After being washed with cold PBS, the cell pellets were resuspended in lysis buffer, which contained 50 µM piperazine-N,N'-bis(2-ethanesulfonic acid)/KOH, pH 6.5, 2 mM EDTA, 0.1% 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 5 mM DTT, 20 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml sodium orthovanadate, and 2 mM PMSF (all purchased from Sigma). The cells were subjected to three freeze/thaw cycles. The lysates were centrifuged at 4°C, and the supernatant fraction was drawn off. Protein concentrations were determined by the Bradford method (Bio-Rad, Hercules, CA). Each sample was loaded with 50 µg of proteins per lane. Proteins were resolved on SDS-PAGE gels, transferred to Immobilon-P polyvinylidene fluoride membranes (Millipore, Bedford, MA). Anti-PKB antibody (Cell Signaling), anti-phosphorylated PKB antibody (Cell Signaling), cIAP1 antibody, cIAP2 antibody, survivin antibody (Santa Cruz Biotechnology, Santa Cruz, CA), Bcl-xl antibody, Bad antibody, phosphorylated Bad antibody (Cell Signaling), and XIAP antibody (Pharmingen/BD Biosciences) had been used separately. The signal was developed using secondary IgG-HRP (Santa Cruz Biotechnology) and SuperSignal Luminol detection solution (Pierce Biotechnology, Rockford, IL). To confirm equal amounts of loaded proteins, the membranes were reprobed with anti--actin monoclonal antibody (Sigma).
Electrophoretic mobility shift assay.
The gel mobility shift assay was carried out using the digoxigenin (DIG) Gel Shift Kit (Roche Molecular Biochemicals). The oligonuclotide used as a probe for the electrophoretic motility shift assay was a double-stranded DNA fragment containing the NF-B consensus sequence 5'-AGTTGAGGGGACTTTCCCAGG-3' (Integrated DNA Technologies, Skokie, IL). Probes were labeled with DIG. Binding reaction mixtures (20 µl) containing 6 µg of nuclear extract protein, 1 µg of poly(dI-dC), 0.1 µg of poly-L-lysine, 2 µl of DIG-labeled oligonucleotide (0.4 µg/µl), and 4 µl of binding buffer were incubated for 15 min at room temperature. The protein-DNA complexes were separated on 6% nondenaturing polyacrylamide gels run at 60 V in 0.5x Tris-borate-EDTA buffer. Transfer was performed for 30 min at 400 mA. After cross-linking, the membrane was washed, incubated for 30 min in blocking buffer, and then incubated for 30 min in antibody solution. The DIG-labeled fragments were visualized by an enzyme immunoassay using anti-DIG-AP, Fab fragments, and the chemiluminescent substrate disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2-(5-chloro)tricyclodecan}-4-yl)phenyl phosphate. The generated chemiluminescent signals were recorded on X-ray film.
Statistics. The data are expressed as means ± SE. Where applicable, ANOVA analyses were performed to evaluate the significance of differences between control and experimental groups. Statistical significance was defined as P < 0.05.
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RESULTS |
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Role of NF-B signaling in delayed neutrophil apoptosis after burn injury.
A potential downstream target of PI3-kinase is NF-
B that has been implicated in survival signaling and has been shown to be activated with thermal injury (28). An EMSA to detect NF-
B activation revealed that activation of the transcription factor was upregulated after burn injury (Fig. 3). The nuclear translocation of NF-
B was assessed via double labeling of RelA and PI in neutrophils. Figure 4 shows that RelA labeling was much more intense in neutrophils from burn-injured rats than in sham samples. RelA labeling in the sham group, although much less intense than in the burn group samples, was inside the nucleus, and the addition of wortmannin to these samples resulted in an extranuclear distribution of RelA. In the absence of wortmannin, burn rat neutrophils showed the presence of most of RelA inside the nucleus. The addition of wortmannin to the burn rat samples clearly attenuated intranuclear RelA distribution. These data indicate that increased translocation of RelA to nucleus with burn injury is significantly attenuated by wortmannin and that activation of NF-
B occurs downstream from PI3-kinase.
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DISCUSSION |
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In our experiment, we found that at an early stage of neutrophil culture (2 h), the delay of apoptosis of PMN from burn-injured rats was dependent on upregulated NF-B activation and increased synthesis of anti-apoptotic factors. However, at a later stage of PMN culture (8 h), the inhibitory effect of thermal injury was not dependent on an increase in synthesis of anti-apoptotic proteins. The phosphorylation of Bad, however, was increased at 8-h neutrophil culture and may be an important factor for PMN survival at this later phase in culture. These data provide information that increased expression of anti-apoptotic Bcl-xl and phosphorylation of Bad, both of which are PI3-kinase-dependent mechanisms for the delay of PMN apoptosis, may operate independently in burn injury. IAP family proteins have been shown to suppress apoptosis induced by a variety of stimuli (13, 34). These proteins directly inhibit activated effector caspases, caspase-3 and caspase-7, and can inhibit the activation of the initiator caspase, caspase-9 (9, 24). Furthermore, it has been reported that IAPs, cIAP1 and cIAP2, bind to TNF receptor-associated factor-1/-2 heterocomplexes and then are recruited to TNF receptors to suppress caspase-8 activation and/or caspase-3 activity (26, 29). IAP family members are inducible by a variety of NF-
B-inducing stimuli, such as TNF, LPS, and G-CSF (7, 33). The findings that proinflammatory stimuli are released into circulation after burn injury could suggest that these stimuli might play roles in inducing IAPs after burn injury. Moreover, cIAP2 expression has been shown to be increased at 1 h after neutrophil culture with G-CSF (13). In our present study, although NF-
B was activated in the freshly isolated and 2-h cultured neutrophils, we failed to demonstrate an effect of burn injury on gene and protein expressions of IAPs in 0- and 1-h cultured neutrophils. These findings suggest that either the IAP-inducing inflammatory stimuli are not expressed at levels required for the activation of IAPs or IAP activation occurs in vivo transiently and then is diminished in freshly isolated PMNs from burn-injured rats. In contrast to the anti-apoptotic activity of NF-
B, it has been shown that NF-
B also promotes apoptosis (17, 42). NF-
B could not only trigger the Fas-death cascade by directly activating the expression of Fas ligand but also regulate gene expression of proapoptotic factors, including Bcl-xs, which antagonizes prosurvival Bcl-2 family members. In our study, we found that at the later stage of neutrophil culture (8 h), NF-
B activity in sham rat PMNs was higher than that in burn-injured rat PMNs and that CHX significantly increased PMN apoptosis in the sham group. This may be related to the proapoptotic effect of NF-
B (such as upregulation of proapoptotic factor expression) on neutrophil apoptosis.
In summary, our results show that delay of neutrophil apoptosis with thermal injury is likely caused by the activation of PI3-kinase-PKB signaling pathway. PI3-kinase-PKB pathway activation in neutrophils after thermal injury occurs upstream of phosphorylation of Bad and NF-B activation. The anti-apoptotic effect of NF-
B with thermal injury depends on increased expression of Bcl-xl, but not IAPs. Moreover, increased Bcl-xl expression and Bad phosphorylation, which are dependent on PI3-kinase activation, appear to operate independently to cause the delay of PMN apoptosis with burn injury.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Brach MA, deVos S, Gruss HJ, and Herrmann F. Prolongation of survival of human polymorphonuclear neutrophils by granulocyte-macrophage colony-stimulating factor is caused by inhibition of programmed cell death. Blood 80: 29202924, 1992.[Abstract]
3. Carter BZ, Milella M, Altieri DC, and Andreeff M. Cytokine-regulated expression of survivin in myeloid leukemia. Blood 97: 27842790, 2001.
4. Castro-Alcaraz S, Miskolci V, Kalasapudi B, Davidson D, and Vancurova I. NF-B regulation in human neutrophils by nuclear I
B
: correlation to apoptosis. J Immunol 169: 39473953, 2002.
5. Chu ZL, McKinsey TA, Liu L, Gentry JJ, Malim MH, and Ballard DW. Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-kappaB control. Proc Natl Acad Sci USA 94: 1005710062, 1997.
6. Cowburn AS, Cadwallader KA, Reed BJ, Farahi N, and Chilvers ER. Role of PI3-kinase-dependent Bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival. Blood 100: 26072616, 2002.
7. Cui X, Imaizumi T, Yoshida H, Tanji K, Matsumiya T, and Satoh K. Lipopolysaccharide induces the expression of cellular inhibitor of apoptosis protein-2 in human macrophages. Biochim Biophys Acta 1524: 178182, 2000.[ISI][Medline]
8. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, and Greenberg ME. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91: 231241, 1997.[CrossRef][ISI][Medline]
9. Deveraux QL and Reed JC. IAP family proteinssuppressors of apoptosis. Genes Dev 13: 239252, 1999.
10. Drost AC, Burleson DG, Cioffi WG, Mason AD, and Pruitt BA Jr. Plasma cytokines after thermal injury and their relationship to infection. Ann Surg 218: 7478, 1993.[ISI][Medline]
11. Frasch SC, Nick JA, Fadok VA, Bratton DL, Worthen GS, and Henson PM. p38 mitogen-activated protein kinase-dependent and -independent intracellular signal transduction pathways leading to apoptosis in human neutrophils. J Biol Chem 273: 83898397, 1998.
12. Fukuda S and Pelus LM. Regulation of the inhibitor-of-apoptosis family member survivin in normal cord blood and bone marrow CD34+ cells by hematopoietic growth factors: implication of survivin expression in normal hematopoiesis. Blood 98: 20912100, 2001.
13. Hasegawa T, Suzuki K, Sakamoto C, Ohta K, Nishiki S, Hino M, Tatsumi N, and Kitagawa S. Expression of the inhibitor of apoptosis (IAP) family members in human neutrophils: up-regulation of cIAP2 by granulocyte colony-stimulating factor and overexpression of cIAP2 in chronic neutrophilic leukemia. Blood 101: 11641171, 2003.
14. Henshall D, Araki T, Schindler C, Lan J, Tiekoter K, Taki W, and Simon R. Activation of Bcl-2-associated death protein and counter-response of Akt within cell populations during seizure-induced neuronal death. J Neurosci 22: 84588465, 2002.
15. Hu Z and Sayeed MM. Suppression of mitochondria-dependent neutrophil apoptosis with thermal injury. Am J Physiol Cell Physiol 286: C170C178, 2004.
16. Karin M and Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-B activity. Rev Immunol 18: 621663, 2000.[CrossRef]
17. Kasibhatla S, Genestier L, and Green DR. Regulation of fas-ligand expression during activation-induced cell death in T lymphocytes via nuclear factor B. J Biol Chem 274: 987992, 1999.
18. Kato T Jr, Delhase M, Hoffmann A, and Karin M. CK2 is a C-terminal IB kinase responsible for NF-
B activation during the UV response. Mol Cell 12: 829839, 2003.[CrossRef][ISI][Medline]
19. Kennedy SG, Wagner AJ, Conzen SD, Jordan J, Bellacosa A, Tsichlis PN, and Hay N. The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev 15: 701713, 1997.
20. Khoshnan A, Tindell C, Laux I, Bae D, Bennett B, and Nel AE. The NF-B cascade is important in Bcl-xL expression and for the anti-apoptotic effects of the CD28 receptor in primary human CD4+ lymphocytes. J Immunol 165: 17431754, 2000.
21. Klein JB, Rane MJ, Scherzer JA, Coxon PY, Kettritz R, Mathiesen JM, Buridi A, and McLeish KR. Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways. J Immunol 164: 42864291, 2000.
22. Kluck RM, Bossy-Wetzel E, Green DR, and Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275: 11321136, 1997.
23. Kucharczak J, Simmons MJ, Fan Y, and Gelinas C. To be, or not to be: NF-B is the answerrole of Rel/NF-
B in the regulation of apoptosis. Oncogene 22: 89618982, 2003.[CrossRef][ISI][Medline]
24. LaCasse EC, Baird S, Korneluk RG, and MacKenzie AE. The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene 17: 32473259, 1998.[CrossRef][ISI][Medline]
25. Lentsch AB and Ward PA. Regulation of experimental lung inflammation. Respir Physiol 128: 1722, 2001.[CrossRef][ISI][Medline]
26. Leo E, Deveraux QL, Buchholtz C, Welsh K, Matsuzawa S, Stennicke HR, Salvesen GS, and Reed JC. TRAF1 is a substrate of caspases activated during tumor necrosis factor receptor-alpha-induced apoptosis. J Biol Chem 276: 80878093, 2001.
27. Minn AJ, Velez P, Schendel SL, Liang H, Muchmore SW, Fesik SW, Fill M, and Thompson CB. Bcl-x(L) forms an ion channel in synthetic lipid membranes. Nature 385: 353357, 1997.[CrossRef][ISI][Medline]
28. Nolan B, Collette H, Baker S, Duffy A, De M, Miller C, and Bankey P. Inhibition of neutrophil apoptosis after severe trauma is NFkappabeta dependent. J Trauma 48: 599604, 2000.[ISI][Medline]
29. Pryhuber GS, Huyck HL, Staversky RJ, Finkelstein JN, and O'Reilly MA. Tumor necrosis factor-a-induced lung cell expression of antiapoptotic genes TRAF1 and cIAP2. Am J Respir Cell Mol Biol 22: 150156, 2000.
30. Savill JS, Wyllie AH, Henson JE, Walport MJ, Henson PM, and Haslett C. Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest 83: 865875, 1989.[ISI][Medline]
31. Senftleben U, Cao Y, Xiao G, Greten FR, Krahn G, Bonizzi G, Chen Y, Hu Y, Fong A, Sun SC, and Karin M. Activation by IKK of a second, evolutionary conserved, NF-
B signaling pathway. Science 293: 14951499, 2001.
32. Shen J, Channavajhala P, Seldin DC, and Sonenshein GE. Phosphorylation by the protein kinase CK2 promotes calpain-mediated degradation of IB
. J Immunol 167: 49194925, 2001.
33. Stehlik C, de Martin R, Binder BR, and Lipp J. Cytokine induced expression of porcine inhibitor of apoptosis protein (IAP) family member is regulated by NF-B. Biochem Biophys Res Commun 243: 827832, 1998.[CrossRef][ISI][Medline]
34. Stehlik C, de Martin R, Kumabashiri I, Schmid JA, Binder BR, and Lipp J. Nuclear factor (NF)-B-regulated X-chromosome-linked IAP gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis. J Exp Med 188: 211216, 1998.
35. Suzuki K, Hasegawa T, Sakamoto C, Zhou YM, Hato F, Hino M, Tatsumi N, and Kitagawa S. Cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis: role in decreased responsiveness to inflammatory cytokines. J Immunol 166: 11851192, 2001.
36. Takeda Y, Watanabe H, Yonehara S, Yamashita T, Saito S, and Sendo F. Rapid acceleration of neutrophil apoptosis by tumor necrosis factor-. Int Immunol 5: 691694, 1993.[Abstract]
37. Tang G, Minemoto Y, Dibling B, Purcell NH, Li Z, Karin M, and Lin A. Inhibition of JNK activation through NF-B target genes. Nature 414: 313317, 2001.[CrossRef][ISI][Medline]
38. Toker A and Cantley LC. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature 387: 673676, 1997.[CrossRef][ISI][Medline]
39. Worthen GS and Henson PM. Mechanisms of acute lung injury. Clin Lab Med 3: 601617, 1983.[ISI][Medline]
40. Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP, and Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275: 11291132, 1997.
41. Zha J, Harada H, Yang E, Jockel J, and Korsmeyer SJ. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 143-3 not BCL-X(L). Cell 87: 619628, 1996.[CrossRef][ISI][Medline]
42. Zheng Y, Ouaaz F, Bruzzo P, Singh V, Gerondakis S, and Beg AA. NF-B RelA (p65) is essential for TNF-
-induced fas expression but dispensable for both TCR-induced expression and activation-induced cell death. J Immunol 166: 49494957, 2001.
43. Zheng Y, Vig M, Lyons J, Van Parijs L, and Beg AA. Combined deficiency of p50 and cRel in CD4+ T cells reveals an essential requirement for nuclear factor B in regulating mature T cell survival and in vivo function. J Exp Med 197: 861874, 2003.
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