Departments of 1Physiology and 2Surgery, and Burn & Shock Trauma Institute, Stritch School of Medicine, Loyola University Medical Center, Maywood, Illinois 60153
Submitted 7 May 2003 ; accepted in final form 8 September 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
burn; rat; polymorphonuclear leukocytes; caspase-3; caspase-9; cytochrome c; Bcl-xL; Bax; Bad; MitoTracker GreenFM; confocal microscopy
Although neutrophils are known to be terminally differentiated cells, several studies have shown that their life span can be modulated (6, 18). The half-life of neutrophils can increase severalfold as they infiltrate into tissue under injury conditions (5, 17). Actions of the gram-negative bacterial product lipopolysaccharide (LPS) and inflammatory mediators such as TNF- and granulocyte-monocyte colony-stimulating factor (GM-CSF), as well as glucocorticoids, have been shown to cause a delay in neutrophil apoptosis (7, 9, 24, 25). Such a delay in apoptosis can prolong neutrophil-mediated tissue damage via their degranulation products, namely, oxidants and proteolytic enzymes (8, 20).
Several investigations have shown that the delay in apoptosis in circulating neutrophils is related to the severity of inflammatory disease (2, 23). Studies have shown delays in apoptosis of pulmonary neutrophils in ARDS (acute respiratory distress syndrome) patients (25) and in neutrophils isolated from the blood of burn-injured and septic patients (5, 30). However, little is known about the mechanism of delay in neutrophil apoptosis in inflammatory/septic conditions.
The delay in cell apoptosis could result from interference with either the mitochondrial pathway or the death receptor pathway of apoptosis, or both (29, 35, 36). Previous studies have examined the role of the death receptor pathway in neutrophil apoptosis (16). However, the role of the mitochondrial pathway of neutrophil apoptosis under sepsis/burn injury conditions has remained unknown. In the mitochondrial pathway, activation of proapoptotic proteins belonging to the Bcl-2 family, such as Bad and Bax, can promote the release of cytochrome c from mitochondria into cytoplasm (1), where it forms a macromolecular complex with the apoptotic protease-activating factor 1 (Apaf-1). The complex cleaves and activates procaspase-9, which in turn causes cleavage and activation of procaspase-3 (14). Caspase-3 is responsible for the breakdown of a number of cytosolic/nuclear proteins resulting in cellular morphological and biochemical changes that are hallmarks of apoptosis, such as cell shrinkage, DNA fragmentation, and chromatin condensation (3, 41). Antiapoptotic proteins also belonging to the Bcl-2 gene family, such as Bcl-2 itself and Bcl-xL, inhibit the release of cytochrome c from mitochondria and thereby prevent the occurrence of apoptosis (38, 40, 45).
In the present study, we have investigated modulations in apoptosis of neutrophils isolated from burn-injured animals and analyzed the potential role of the mitochondrial pathway in such modulations. We have examined mitochondrial morphological changes that are characteristic of apoptosis and assessed the involvement of pro- and antiapoptotic Bcl-2 family members that could be implicated in the mitochondrial pathway of apoptosis.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation and culturing of neutrophils. Blood (1012 ml) was collected through cardiac puncture into heparinized syringes. Neutrophils were isolated from heparinized blood via Ficoll-Paque (Pharmacia, Pea Pack, NJ) gradient centrifugation. The erythrocyte/granulocyte pellet was diluted with normal saline (1:1). Erythrocytes were sedimented in 3% dextran (Sigma, St. Louis, MO) for 1 h at room temperature; the resulting supernatant was collected and centrifuged for 10 min at 4°C. Red blood cells remaining in the pellet were suspended and 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 (FCS) (Cellgro Mediatech), 100 U/ml penicillin, 100 µg/ml streptomycin, and 300 µg/ml glutamine (GIBCO BRL, Grand Island, NY). Neutrophil preparation routinely contained 95% neutrophils as identified by the Giemsa stain. Neutrophils (5x106) were put into each well of six-well plates (Fisher Scientific, Pittsburgh, PA), which were then incubated in 5% CO2-95% humidified air at 37°C for 2 or 8 h.
Determination of neutrophil apoptosis. Neutrophil apoptosis was measured by using the annexin V-fluorescein isothiocyanate (FITC)/PI apoptosis assay kit (Pharmingen, BDSciences) and flow cytometry. The experiment was performed by following the manufacturer's instructions, with minor changes. Briefly, after isolation or incubation for 2 or 8 h, neutrophils were washed twice with ice-cold PBS and then resuspended in binding buffer. Annexin V-FITC and PI were added into the culture tube. 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 staining but negative for PI staining. Cells positive for both annexin V and PI staining were considered necrotic cells. Cell survival/apoptosis was expressed as a percentage of neutrophils relative to the total number of counted neutrophils.
Determination of mitochondrial morphology. For the staining of neutrophil mitochondria alone, 105 freshly prepared or cultured neutrophils were incubated in 500 µl of modified RPMI 1640 with 200 nM MitoTracker GreenFM for 30 min at 37°C in an atmosphere of 5% CO2. Neutrophils were layered on glass microscope slides by using cytospin in the dark. The slides were air dried, and neutrophils were examined via confocal laser scanning microscope (Bio-Rad Laboratories, Hercules, CA). Numbers of neutrophils with differing mitochondrial staining patterns were counted in the microscope field and expressed as percentages of the total number of counted neutrophils.
The simultaneous staining of mitochondria and active caspase-3 in the neutrophil was performed according to a method described by Maianski and coworkers (24), with minor modifications. Briefly, 106 freshly isolated or cultured neutrophils were washed once in ice-cold PBS and stained with 2 µM MitoTracker GreenFM. Neutrophils were then fixed with 2% (wt/vol) paraformaldehyde in PBS for 15 min at room temperature, washed twice in PBS, and resuspended in staining buffer containing 0.1% saponin (wt/vol) and 1% (wt/vol) bovine serum albumin (BSA) (Sigma) in PBS. Permeabilized neutrophils were incubated with antibody against active caspase-3 (Cell Signaling, Beverly, MA) or resuspended in buffer without the antibody (negative control). After incubation, neutrophils were washed twice and resuspended in secondary antibody (Alexa 488-conjugated goat anti-mouse IgG; Molecular Probes, Eugene, OR) at a final concentration of 5.0 µg/ml. Neutrophils on the slides, prepared by using cytospin, were examined under a confocal laser scanning microscope as described above.
RT-PCR. Total RNA was isolated from the neutrophils by using TRI Reagent according to the manufacturer's instructions (Molecular Research Center, Cincinnati, OH). Neutrophils were washed twice with ice-cold PBS and suspended in lysis medium for 5 min, after which chloroform was added. After centrifugation, the upper aqueous phase was collected and precipitated by adding isopropanol. The resulting RNA pellet was separated after centrifugation and washed with 70% ethanol.
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 transcriptase (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 samples were denatured at 95°C for 15 min and amplified for 30 cycles (rat GAPDH, or RGAPDH) or 32 cycles (A1, Mcl-1, Bcl-xL, Bad, Bax), with the last cycle extended at 72°C for 10 min. Samples were then resolved on a 2% agarose gel and visualized with ethidium bromide.
The following six primers were used: rat A1 (forward: 5'-TGTATATCCACTCCCTGGCTG-3', reverse: 5'-AGTCACAATCCTTCCCCAGTT-3'); rat Mcl-1 (forward: 5'-TGGACATTAAAAACGAGGACG-3', reverse: 5'-AAGAACTCCACAAACCCATCC-3'); rat Bcl-xL (forward: 5'-TGGAAAGCGTAGACAAGGAGA-3', reverse: 5'-AGTGAGCCCAGCAGAACTACA-3'); rat Bax (forward: 5'-AAGACAGGGGCCTTTTTGTTA-3', reverse: 5'-GAAGTTGCCATCAGCAAACAT-3'); rat Bad (forward: 5'-TGTTCCAGATCCCAGAGTTTG, reverse: 5'-CTCCATCCCTTCATCTTCCTC-3'); and RGAPDH (forward: 5'-CCATCACCATCTTCCAGGAG-3', reverse: 5'-CCTGCTCACCACCTTCTTG-3').
Western blotting analyses. Separation of neutrophil mitochondrial and cytosolic fractions was performed for the determination of cytochrome c release (procedure described in Ref. 13). The freshly isolated or cultured neutrophils were rinsed with cold PBS twice and then homogenized in cold suspension buffer [20 mM HEPES-KOH (pH 7.5), 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml aprotinin, 10 µg/ml leupeptin, 5 µg/ml pepstatin, and 12.5 µg/ml N-acetyl-Leu-Leu-norleucine]. Homogenates were centrifuged at 750 g for 10 min at 4°C. Resulting supernatants were centrifuged at 8,000 g for 20 min at 4°C. The 8,000 g pellets were used as the mitochondrial fraction. The 8,000 g supernatants were further centrifuged at 100,000 g for 60 min at 4°C, and resulting supernatants were used as the cytosolic fraction.
The whole neutrophil lysates were obtained as follows. After being washed with cold PBS, the neutrophil pellets were resuspended in lysis buffer that contained 50 µM PIPES-KOH (pH 6.5), 2 mM EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, 5 mM DTT, 20 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml sodium orthovanadate, and 2 mM PMSF (Sigma). The cell lysates were subjected to three freeze/thaw cycles and then centrifuged at 4°C. The supernatant fraction was drawn off, and its protein concentration was determined using the Bradford method (Bio-Rad). Each sample was loaded with 50 µg of protein per lane. Proteins were resolved on SDS-PAGE gels and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA). Anticaspase-3 antibody (Cell Signaling), anti-caspase-9 antibody (Cell Signaling), anti-cytochrome c antibody, anti-Mcl-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bax antibody, anti-Bcl-xL antibody, anti-Bad antibody, and anti-phosphorylated Bad antibody (Cell Signaling) were employed in separate experiments. The membrane was incubated with appropriate antibody conjugated with horse-radish peroxidase (Santa Cruz Biotechnology). The signal was developed by using Super-Signal Luminol detection solution (Pierce Biotechnology, Rockford, IL). To confirm an equal amount of loaded proteins, the membranes were reprobed with anti--actin monoclonal antibody (Sigma).
Statistics. Data are expressed as means ± SE. Where applicable, the Student's t-test was used to evaluate the significance of differences between burn and sham group sample means. Statistical significance was defined as P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Expression of Bcl-2-related genes and proteins in neutrophils. Figure 2 shows representative mRNA expression and densitometric analysis (mean ± SE values of ratios to RGAPDH mRNA) of antiapoptotic members of the Bcl-2 family, Mcl-1, A1 and Bcl-xL, as well as proapoptotic Bax and Bad. The expression levels of Mcl-1 and A1 were not significantly different between freshly prepared or cultured sham and burn rat neutrophils. However, mRNA expression of antiapoptotic Bcl-xL showed an increase in freshly isolated and 2-h-cultured PMNs of burn rats compared with sham rats. At 8 h of incubation, burn rat PMN Bcl-xL mRNA expression was lower than in the sham group. Expression of either Bax or Bad mRNA expression in burn rat PMNs was not different from that in the sham group at 0 or 2 h of incubation but was decreased at 8 h of incubation compared with sham rat PMNs. The decrease in Bax and Bad mRNA in burn rat PMNs at 8 h of incubation coincided with the decrease in Bcl-xL mRNA expression. The Western blots of antiapoptotic and proapoptotic proteins and densitometric analyses are shown in Fig. 3. Mcl-1 protein level was not different between sham and burn rat neutrophils. Bcl-xL protein level was apparently higher in both freshly isolated and 2-h-incubated PMNs from rats subjected to burn injury than in the sham group. However, after 8 h of incubation, PMNs from burn rats showed decreased protein levels of Bcl-xL compared with sham. This pattern of change in Bcl-xL protein in burn rat PMNs was similar to changes observed in Bcl-xL mRNA of burn rat PMNs. Although Bax mRNA was found to be decreased (compared with sham group) only at 8-h-incubated PMNs, the Bax protein level was evidently decreased in freshly isolated as well as both 2- and 8-h-incubated PMNs from burned rats, compared with sham. To investigate the role of proapoptotic Bad protein, we assessed both total Bad protein contents as well as levels of phosphorylated Bad. The proapoptotic role of Bad depends on its dephosphorylation, because dephosphorylated Bad sequesters Bcl-xL, leading to mitochondrial membrane destabilization, release of cytochrome c from mitochondria, and apoptosis (46). The total quantity of Bad protein in PMNs was apparently not affected with burn injury. The quantity of phosphorylate Bad was not detected in freshly isolated PMN from burn or sham groups but was found to increase in cultured PMNs of both sham and burn groups. However, the increase in phosphorylated Bad at 8 h was substantially greater in the burn group than in the sham group. The increase in phosphorylated Bad would be associated with decreased dephosphorylation of Bad and thus with an attenuation in apoptosis.
|
|
Mitochondrial structure changes associated with apoptosis. The MitoTracker GreenFM dye, which has high affinity and specificity for lipid membranes in mitochondria and produces monofluorescent green images, was employed to examine mitochondrial membrane morphology (Fig. 4). Previous studies have shown that surviving neutrophils exhibited a "tubular" structure of mitochondria; on the other hand, nonsurviving neutrophils exhibited an "aggregation" of mitochondria (24). There was apparently no distinguishable difference in the configuration of mitochondria in the freshly isolated neutrophils (0 h) from sham and burn-injured rats. In both groups, 95% (±3%) of the neutrophils showed the tubular configuration in the mitochondria. After 2 h of incubation, the mitochondria in 16% (±2%) of sham rat neutrophils showed aggregations of mitochondria, whereas 5% (±1%) of burned rat PMNs showed the aggregated morphology. At 8 h of incubation, there appeared to be a further loss of the tubular configuration and appearance of aggregation of mitochondria in 35% (±2%) of sham neutrophils, whereas 11% (±2%) of neutrophils from burn rats exhibited aggregated structures.
|
Cytochrome c release from mitochondria. Figure 5 shows representative Western blots and densitometric analyses of cytochrome c levels in cytosolic and mitochondrial fractions of burn and sham rat PMNs. Cytochrome c was present as a single band representing a molecular mass of 15 kDa. In both freshly isolated and cultured neutrophils, cytochrome c was lower in the cytosolic fraction and higher in the mitochondrial fraction in the burn group than in the sham group. These results indicate a decreased release of cytochrome c from mitochondria into cytosol in the burn rat PMNs compared with the sham group.
|
Activation of caspase-9 and -3. Figure 6 shows representative blots of cleaved caspase-9 and caspase-3 and densitometric analyses of the blots. There were measurable quantities of active caspase-9 in freshly isolated neutrophils. However, the amount of active caspase-9 in sham rat neutrophils was greater than in burn-injured rat neutrophils. After sham rat neutrophils were incubated for 2 or 8 h, there were greater quantities of the cleavage products of caspase-9 compared with the burn groups. Caspase-3 activation showed similar changes as observed in caspase-9 activation with burn injury. Caspase-3 activation was clearly lower in burn rat PMNs than in sham.
|
Simultaneous staining of mitochondria and caspase-3 activation in neutrophils. Neutrophils were simultaneously stained with mitochondria and cleaved caspase-3. Confocal microscopic analysis showed that freshly prepared neutrophils from sham and burn-injured rats did not have a strongly detectable staining for the active caspase-3. In 2-h cultured sham rat neutrophils, there was clearly demonstrable staining for active caspase-3 along with the appearance of the aggregated configuration of mitochondria (Fig. 7A), but in 2-h cultured neutrophils from burn-injured rats, there was no appearance of aggregated mitochondria and a barely demonstrable staining of caspase-3 activation (Fig. 7B). In 8-h cultured neutrophils from both group rats, mitochondrial structure changes and caspase-3 activation were detectable, but the active caspase-3 staining showed positive in only a few 8-h cultured burn-injured rat neutrophils, whereas a large number of the sham rat neutrophils demonstrated a strong positive staining with cleaved caspase-3 (Fig. 7, C and D). Western blotting and confocal laser scanning microscopic analysis of caspase-3 activation were in agreement in indicating that burn injury probably caused an inhibition of caspase-3 activation and that the caspase-3 inhibition was likely accompanied by a decreased incidence of mitochondrial morphological changes associated with apoptosis.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Whereas neutrophil apoptosis is critical to the resolution of inflammation, a delay in apoptosis of neutrophils could prolong their production and release of proinflammatory mediators that would be detrimental to the host (4). The delay in apoptosis also may be beneficial by providing for a prolongation of host defense against pathogens. Thus a mere delay of apoptosis in inflammatory conditions such as burn injury may be detrimental as well as beneficial to the host. In previous studies from our laboratory (11, 34), we showed that in the rat burn injury model, similar to that employed in this study, neutrophils are hyperactivated and contribute to tissue injury. The hyperactivation of neutrophils was evident in their increased level of production, which was in turn related to tissue damage and dysfunction in the intestine (12, 39). Thus the delay in apoptosis and hyperaction of PMNs with thermal injury, as observed in this and the previous studies, would appear to contribute to tissue damage and organ dysfunction.
Data presented here show that the delay in neutrophil apoptosis with burn injury was accompanied by reciprocal changes in survival of cells. The delay in apoptosis with burn also accompanied a decrease in the proportion of necrotic cells at 8 h of incubation, whereas necrotic cells were 18.9% in the sham groups and 5.3% in the burn groups. The decrease in the proportion of necrotic cells with burn likely represents a decrease in apoptotic cell death contributing to fewer apoptotic cells eventually undergoing necrosis.
Mitochondria have been shown to initiate apoptosis by releasing factors, such as cytochrome c, which participate in the formation of the apoptosome complex that activates caspase-9/-3 cascade. The involvement of mitochondria in apoptosis has been assessed via measurements such as mitochondrial permeability transition (MPT), mitochondrial membrane potential, and morphological changes in mitochondria (14, 24, 44). In the present study, assessments of mitochondrial structural changes supported the hypothesis that burn injury inhibited or prevented a breakdown of PMN mitochondrial integrity that accompanies apoptosis (31). It should be pointed out that freshly isolated neutrophils from sham and burn-injured rats did not show a discernible difference in mitochondrial morphology, but we did find a lesser degree of release of cytochrome c from mitochondria in freshly isolated burn rat PMNs compared with PMNs from sham rats. It is likely that mitochondrial morphology change may occur later than the leakage of cytochrome c from mitochondria.
The roles of antiapoptotic and proapoptotic factors have not been previously studied in detail under burn injury conditions. It is known that proapoptotic and antiapoptotic members of the Bcl-2 family modulate cell death through their ability to form complex homodimers and heterodimers that ultimately influence the insertion of Bax and Bax-like proapoptotic proteins into the outer mitochondrial membrane (28, 31, 37, 42, 43). The insertion of Bax into the mitochondrial membrane has been shown to trigger the release of cytochrome c and cell apoptosis (14, 19, 22). Previous studies have also shown that whereas the antiapoptotic protein Bcl-2 itself is not expressed in PMNs (10, 14, 26), other antiapoptotic Bcl-2 family members might be involved in delay of PMN apoptosis induced by GM-CSF and other proinflammatory mediators (16, 24, 27). In the present study, we did not detect a difference in antiapoptotic Mcl-1 or A1 but did find differences in Bax, Bad, and Bcl-xL expression between sham and burn-injured rat neutrophils. In freshly isolated PMNs or in PMNs after 2 h of incubation, the delay in neutrophil apoptosis with burn injury could be partly due to increased expression of antiapoptotic Bcl-xL. Bcl-xL has been shown to stabilize mitochondrial membrane and block cytochrome c release from mitochondria (38, 45). Our measurement of decreased cytochrome c release from mitochondria occurring in burn rat PMNs at 0 and 2 h of incubations coincided with the increased Bcl-xL mRNA and protein expression. Although Bax mRNA was not affected in burn rat PMNs at 0 or 2 h of incubation, Bax protein level in burn rat PMNs was decreased at 0 or 2 h of incubation. This could be due to either a change in Bax protein synthesis or its degradation in the face of unaffected Bax gene expression. Thus the burn-related decrease in Bax protein at 0 and 2 h of PMN incubation could contribute to PMN apoptosis delay due to a decrease in its incorporation into mitochondrial membrane (45). At8hof incubation, burn rat PMNs, compared with those of the sham group, showed a decrease in Bax protein while Bcl-xL protein also paradoxically appeared to decrease. This finding suggests an independent role of decreased Bax in contributing to mitochondrial membrane stability in 8-h-incubated burn rat PMNs. Our data concerning phosphorylated Bad levels are also indicative of its potential role in apoptosis delay with burn injury observed in PMNs incubated for 8 h. The observed increase in phosphorylated Bad in 8-h-incubated PMNs from burn-injured rats implies a decrease in dephosphorylation of Bad and consequent decrease in apoptosis relative to corresponding sham rat PMNs. Thus the decrease in apoptosis in 8-h-incubated burn rat PMNs may be a result of multiple factors. Not only a decrease in Bax but also an increase in phosphorylated Bad could cause the delay in PMN apoptosis via stabilization of mitochondrial membrane, even in the face of the paradoxical decrease in Bcl-xL.
We found a detectable level of active caspase-9 in freshly isolated sham rat neutrophils. Active caspase-9 was, however, attenuated in burn rat PMNs. Because caspase-9 reflects activation of the mitochondrial pathway of apoptosis, the burn-related changes in caspase-9 activation support the role of suppression of the mitochondrial pathway in PMN apoptosis delay with burn. Caspase-3 activation is an event distal to both the death receptor and mitochondria-related apoptosis pathways; its attenuated activation in burn rat PMNs implicates a suppression of both the death receptor and/or mitochondrial pathway contributing to PMN survival.
In summary, the present study definitively demonstrates that a suppression of mitochondrial (intrinsic) pathway contributes to the delay in neutrophil apoptosis with burn injury. The data also demonstrate that both a decrease in the critical proapoptotic factors (Bax and dephosphorylated Bad) and an increase in antiapoptotic factors (Bcl-xL) play a role in the modulation of the mitochondrial caspase-9 cascade to produce delay in PMN apoptosis after thermal injury.
![]() |
ACKNOWLEDGMENTS |
---|
GRANTS
This work was supported by National Institute of General Medical Sciences Grants R01-GM-56865 and R01-GM-52325.
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Akgul C, Moulding DA, and Edwards SW. Molecular control of neutrophil apoptosis. FEBS Lett 487: 318322, 2001.[CrossRef][ISI][Medline]
3. Alnemri ES. Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases. J Cell Biochem 64: 3342, 1997.[CrossRef][ISI][Medline]
4. Biffl WL, Moore EE, Zallen G, Johnson JL, Gabriel J, Offner PJ, and Silliman CC. Neutrophils are primed for cytotoxicity and resist apoptosis in injured patients at risk for multiple organ failure. Surgery 126: 198202, 1999.[CrossRef][ISI][Medline]
5. Chitnis D, Dickerson C, Munster AM, and Winchurch RA. Inhibition of apoptosis in polymorphonuclear neutrophils from burn patients. J Leukoc Biol 59: 835839, 1996.[Abstract]
6. Colotta F, Re F, Polentarutti N, Sozzani S, and Mantovani A. Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products. Blood 80: 20122020, 1992.[Abstract]
7. Cox G. Glucocorticoid treatment inhibits apoptosis in human neutrophils. Separation of survival and activation outcomes. J Immunol 154: 47194725, 1995.
8. Cox G. IL-10 enhances resolution of pulmonary inflammation in vivo by promoting apoptosis of neutrophils. Am J Physiol Lung Cell Mol Physiol 271: L566L571, 1996.
9. Das S, Bhattacharyya S, Ghosh S, and Majumdar S. TNF-alpha induced altered signaling mechanism in human neutrophil. Mol Cell Biochem 197: 97108, 1999.[CrossRef][ISI][Medline]
10. Epling-Burnette PK, Zhong B, Bai F, Jiang K, Bailey RD, Garcia R, Jove R, Djeu JY, Loughran TP Jr, and Wei S. Cooperative regulation of Mcl-1 by Janus kinase/stat and phosphatidylinositol 3-kinase contribute to granulocyte-macrophage colony-stimulating factor-delayed apoptosis in human neutrophils. J Immunol 166: 74867495, 2001.
11. Fazal N, Al-Ghoul WM, Schmidt MJ, Choudhry MA, and Sayeed MM. Lyn- and ERK-mediated vs. Ca2+-mediated neutrophil O responses with thermal injury. Am J Physiol Cell Physiol 283: C1469C1479, 2002.
12. Fazal N, Shamim M, Khan SS, Gamelli RL, and Sayeed MM. Neutrophil depletion in rats reduces burn-injury induced intestinal bacterial translocation. Crit Care Med 28: 15501555, 2000.[ISI][Medline]
13. Fujimura M, Morita-Fujimura Y, Murakami K, Kawase M, and Chan PH. Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats. J Cereb Blood Flow Metab 18: 12391247, 1998.[ISI][Medline]
14. Green DR and Reed JC. Mitochondria and Apoptosis. Science 281: 13091312, 1998.
15. Henson PM and Johnston RB. Tissue injury in inflammation: oxidants, proteinases and cationic proteins. J Clin Invest 79: 669674, 1988.[ISI]
16. Iwai K, Miyawaki T, Takizawa T, Konno A, Ohta K, Yachie A, Seki H, and Taniguchi N. Differential expression of bcl-2 and susceptibility to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes, and neutrophils. Blood 84: 12011208, 1994.
17. Jimenez MF, Watson RW, Parodo J, Evans D, Foster D, Steinberg M, Rotstein OD, and Marshall JC. Dysregulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome. Arch Surg 132: 12631269, 1997.[Abstract]
18. Keel M, Ungethum U, Steckholzer U, Niederer E, Hartung T, Trentz O, and Ertel W. Interleukin-10 counterregulates proinflammatory cytokine-induced inhibition of neutrophil apoptosis during severe sepsis. Blood 90: 33563363, 1997.
19. 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.
20. Lee A, Whyte MKB, and Haslett C. Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediators. J Leukoc Biol 54: 283288, 1993.[Abstract]
21. Lehrer RI, Ganz T, Selsted ME, Babior BM, and Curnutte JT. Neutrophils and host defense. Ann Intern Med 109: 127142, 1988.[ISI][Medline]
22. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, and Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: 479489, 1997.[ISI][Medline]
23. Mahidhara R and Billiar TR. Apoptosis in sepsis. Crit Care Med 4, Suppl: N105N113, 2000.[CrossRef]
24. Maianski NA, Mul FP, van Buul JD, Roos D, and Kuijpers TW. Granulocyte colony-stimulating factor inhibits the mitochondria-dependent activation of caspase-3 in neutrophils. Blood 99: 672679, 2002.
25. Matute-Bello G, Liles WC, Radella 2nd F, Steinberg KP, Ruzinski JT, Jonas M, Chi EY, Hudson LD, and Martin TR. Neutrophil apoptosis in the acute respiratory distress syndrome. Am J Respir Crit Care Med 156: 19691977, 1997.
26. Moulding DA, Akgul C, Derouet M, White MR, and Edwards SW. BCL-2 family expression in human neutrophils during delayed and accelerated apoptosis. J Leukoc Biol 70: 783792, 2001.
27. Moulding DA, Quayle JA, Hart CA, and Edwards SW. Mcl-1 expression in human neutrophils: regulation by cytokines and correlation with cell survival. Blood 92: 24952502, 1998.
28. Murphy KM, Streips UN, and Lock RB. Bcl-2 inhibits a Fas-induced conformational change in the Bax N terminus and Bax mitochondrial translocation. J Biol Chem 275: 1722517228, 2000.
29. Nagata S. Apoptosis by death factor. Cell 88: 355365, 1997.[ISI][Medline]
30. Nolan B, Collette H, Baker S, Duffy A, De M, Miller C, and Bankey P. Inhibition of neutrophil apoptosis after severe trauma is NFB dependent. J Trauma 48: 599604, 2000.[ISI][Medline]
31. Nomura M, Shimizu S, Ito T, Narita M, Matsuda H, and Tsujimoto Y. Apoptotic cytosol facilitates Bax translocation to mitochondria that involves cytosolic factor regulated by Bcl-2. Cancer Res 59: 55425548, 1999.
32. Savill J. Apoptosis in resolution of inflammation. J Leukoc Biol 61: 375380, 1997.[Abstract]
33. 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 macrophage. J Clin Invest 83: 865875, 1989.[ISI][Medline]
34. Sayeed MM. Neutrophil signaling alteration: an adverse inflammatory response after burn shock. Medicina (B Aires) 58: 386392, 1998.[ISI][Medline]
35. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, and Peter ME. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17: 16751687, 1998.
36. Scaffidi C, Schmitz I, Zha J, Korsmeyer SJ, Krammer PH, and Peter ME. Differential modulation of apoptosis sensitivity in CD95 type I and type II cells. J Biol Chem 274: 2253222538, 1999.
37. Sedlak TW, Oltvai ZN, Yang E, Wang K, Boise LH, Thompson CB, and Korsmeyer SJ. Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proc Natl Acad Sci USA 92: 78347838, 1995.[Abstract]
38. Simonen M, Keller H, and Heim J. The BH3 domain of Bax is sufficient for interaction of Bax with itself and with other family members and it is required for induction of apoptosis. Eur J Biochem 249: 8591, 1997.[Abstract]
39. Sir O, Fazal N, Choudhry MA, Goris RJ, Gamelli RL, and Sayeed MM. Role of neutrophils in burn-induced microvascular injury in the intestine. Shock 14: 113117, 2000.[ISI][Medline]
40. Tan Y, Demeter MR, Ruan H, and Comb MJ. BAD Ser-155 phosphorylation regulates BAD/Bcl-XL interaction and cell survival. J Biol Chem 275: 2586525869, 2000.
41. Thornberry NA and Lazebnik Y. Caspases: enemies within. Science 281: 13121316, 1998.
42. Xiang J, Chao DT, and Korsmeyer SJ. BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc Natl Acad Sci USA 93: 1455914563, 1996.
43. 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.
44. Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin SA, Petit PX, Mignotte B, and Kroemer G. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 182: 367377, 1995.[Abstract]
45. Zha H, Aime-Sempe C, Sato T, and Reed JC. Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2. J Biol Chem 271: 74407444, 1996.
46. 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.[ISI][Medline]