Rate Of Neutrophil Apoptosis Research Articles

Neutrophils recruited to the site ofMycobacterium bovisBCG infection undergo apoptosis and modulate lipid body biogenesis and prostaglandin E2production by macrophages

Neutrophil influx to sites of mycobacterial infections is one of the first events of tuberculosis pathogenesis. However, the role of early neutrophil recruitment in mycobacterial infection is not completely understood. We investigated the rate of neutrophil apoptosis and the role of macrophage uptake of apoptotic neutrophils in a pleural tuberculosis model induced by BCG. Recruited neutrophils were shown to phagocyte BCG and a large number of neutrophils undergo apoptosis within 24 h. Notably, the great majority of apoptotic neutrophils were infected by BCG. Increased lipid body (lipid droplets) formation, accompanied by prostaglandin E(2) (PGE(2)) and TGF-beta1 synthesis, occurred in parallel to macrophage uptake of apoptotic cells. Lipid body and PGE(2) formation was observed after macrophage exposure to apoptotic, but not necrotic or live neutrophils. Blockage of BCG-induced lipid body formation significantly inhibited PGE(2) synthesis. Pre-treatment with the pan-caspase inhibitor zVAD inhibited BCG-induced neutrophil apoptosis and lipid body formation, indicating a role for apoptotic neutrophils in macrophage lipid body biogenesis in infected mice. In conclusion, BCG infection induced activation and apoptosis of infected neutrophils at the inflammatory site. The uptake of apoptotic neutrophils by macrophages leads to TGF-beta1 generation and PGE(2)-derived lipid body formation, and may have modulator roles in mycobacterial pathogenesis.

Reactive Oxygen Species Regulate Neutrophil Recruitment and Survival in Pneumococcal Pneumonia

The role of NADPH oxidase activation in pneumonia is complex because reactive oxygen species contribute to both microbial killing and regulation of the acute pulmonary infiltrate. The relative importance of each role remains poorly defined in community-acquired pneumonia. We evaluated the contribution of NADPH oxidase-derived reactive oxygen species to the pathogenesis of pneumococcal pneumonia, addressing both the contribution to microbial killing and regulation of the inflammatory response. Mice deficient in the gp91(phox) component of the phagocyte NADPH oxidase were studied after pneumococcal challenge. gp91(phox)(-/-) mice demonstrated no defect in microbial clearance as compared with wild-type C57BL/6 mice. A significant increase in bacterial clearance from the lungs of gp91(phox)(-/-) mice was associated with increased numbers of neutrophils in the lung, lower rates of neutrophil apoptosis, and enhanced activation. Marked alterations in pulmonary cytokine/chemokine expression were also noted in the lungs of gp91(phox)(-/-) mice, characterized by elevated levels of tumor necrosis factor-alpha, KC, macrophage inflammatory protein-2, monocyte chemotactic protein-1, and IL-6. The greater numbers of neutrophils in gp91(phox)(-/-) mice were not associated with increased lung injury. Levels of neutrophil elastase in bronchoalveolar lavage were not decreased in gp91(phox)(-/-) mice. During pneumococcal pneumonia, NADPH oxidase-derived reactive oxygen species are redundant for host defense but limit neutrophil recruitment and survival. Decreased NADPH oxidase-dependent reactive oxygen species production is well tolerated and improves disease outcome during pneumococcal pneumonia by removing neutrophils from the tight constraints of reactive oxygen species-mediated regulation.

Phospho- AKT Is Not Sufficient To Delay Neutrophil Apoptosis.

Neutrophils play an important role in the innate immune response and usually are the first cells to defend the organism against infections. The lifetime of neutrophils is tightly regulated and once the mature cells left the bone marrow most of them undergo apoptosis within 48 hours. The rate of neutrophil apoptosis can be modulated by a variety of cytokines, including granulocyte colony-stimulating factor (G-CSF). To investigate the involvement of the G-CSF receptor in the neutrophil susceptibility to apoptosis we utilized mice bearing truncation mutations on the G-CSF receptor (G-CSFR) and also the G:EpoR mutation, where the entire cytoplasmic (signaling) domain of the G-CSFR is replaced with that of the erythropoietin receptor (EpoR). We examined the role of AKT-mTOR pathway in neutrophil apoptosis. Bone marrow neutrophils from wild type (C57BL/6) and mutant mice where cultured in presence or absence of G-CSF and/or Rapamycin (a specific mTor inhibitor); the apoptosis and the level of phosphorylation of AKT and ERK were evaluated. Neutrophils extracted from G:EpoR mice do not respond to G-CSF treatment and undergo apoptosis within 24 hours, although WT neutrophils can be partially protected against apoptosis when treated with G-CSF. Interestingly, G-CSF treatment leads to activation of Akt and Erk kinases, even in G:EpoR neutrophils. Furthermore, the addition of Rapamycin had no effect in the apoptosis of WT neutrophils in presence or absence of G-CSF. To further elucidate the role of the AKT-mTOR pathway, Sca+Lin- WT bone marrow cells were utilized in an in vitro G-CSF dependent granulocytic differentiation assay. The experiments showed that the mTor pathway is important for the early stages of myeloid differentiation but has little influence in the late stages, and also in the life span of neutrophils. Interestingly, over expression of a constitutively active form of AKT (myr-AKT) in Sca+Lin- cells impaired proliferation even though the differentiation was not affected. To overcome this, a tetracycline response element (TRE) conditional expression system was utilized. These experiments showed that myr-AKT expression does not rescue neutrophils from apoptosis. In conclusion, our results indicate that after treatment with G-CSF; AKT and ERK are phosphorylated in WT and G:EpoR neutrophils, despite this, G-CSF does not suppress G:EpoR neutrophil apoptosis while WT neutrophils can have their apoptosis partially delayed. We also found that mTor is involved in the early stages of G-CSF dependent myeloid differentiation and its inhibition can partially arrest differentiation. Conversely, rapamycin does not affect neutrophil apoptosis, in presence or not of G-CSF. In concordance with these results overexpression of a constitutive active form of AKT, myr-AKT does not rescue neutrophils from apoptosis and it is also detrimental to early stages of granulocytic proliferation. These data strongly suggest that the AKT-mTor pathway while important in early myeloid differentiation and proliferation does not play a role in cytokine suppression of neutrophil apoptosis.

Apoptosis of human neutrophils induced by protein phosphatase 1/2A inhibition is caspase‐independent and serine protease‐dependent

Protein phosphatase (PP) activity is associated with the regulation of apoptosis in neutrophils. However, the underlying regulatory mechanism(s) in apoptosis remain unclear. The type of cell death induced by okadaic acid (OA), the inhibitor of PP1 and PP2A, is characterized by apoptotic morphological changes of the cells and annexin V-positive staining without DNA fragmentation. The apoptotic effects of OA and calyculin A on neutrophils were observed at concentrations ranging from 50 to 200 nM, or 10 to 50 nM, respectively. Cyclosporine A (a PP2B specific inhibitor), however, did not exhibit any pro-apoptotic effects. OA and calyculin A, but not cyclosporine A, exhibited significant effects on protein levels and on the electrophoretic mobility of Mcl-1. zVAD-fmk, a pancaspase inhibitor, failed to inhibit the effect of OA on the caspase-3 activity, procaspase-3 processing, and the apoptotic rate of neutrophils. However, 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF), a general serine protease inhibitor, significantly abrogated the OA-induced mobility shift in procaspase-3, caspase-3 activation, and the apoptotic morphological changes in neutrophils. Moreover, OA enhanced the serine protease activity of the neutrophils. The addition of the proteinase-3 protein increased the rate of neutrophil apoptosis, which was also blocked by AEBSF but not by zVAD-fmk. These results suggest that OA induces procaspase-3 processing but that OA-induced apoptosis is caspase-independent and serine protease-dependent.

Comparative Analysis of the Abilities of Shiga Toxins 1 and 2 To Bind to and Influence Neutrophil Apoptosis

Hemolytic-uremic syndrome (HUS), the life-threatening complication following infection by the intestinal pathogen Escherichia coli O157:H7, is due to the ability of the pathogen to produce toxins in the Shiga toxin (Stx) family. Activated neutrophils are observed in HUS patients, yet it is unclear whether Stx exerts a direct effect on neutrophils or whether the toxin acts indirectly. The effect of Stx1 and Stx2 on human neutrophils was examined. Neither Stx1 nor Stx2 altered the rate of neutrophil apoptosis. Minimal binding of either toxin to neutrophils was observed, and the toxin was easily eluted from the cells. Stx1 and Stx2 were found to circulate in the plasma of mice following intravenous injection, and both toxins were cleared rapidly from the blood. Together these results suggest that neither Stx1 nor Stx2 interacts directly with neutrophils.

Aminopeptidase N (CD13) Regulates Tumor Necrosis Factor-α-induced Apoptosis in Human Neutrophils

Neutrophil apoptosis plays a central role in the resolution of granulocytic inflammation. We have shown previously that tumor necrosis factor-alpha (TNFalpha) enhances the rate of neutrophil apoptosis at early time points via a mechanism involving both TNF receptor (TNFR) I and TNFRII. Here we reveal a marked but consistent variation in the magnitude of the pro-apoptotic effect of TNFalpha in neutrophils isolated from healthy donors, and we show that inhibition of cell surface aminopeptidase N (APN) using actinonin, bestatin, or inhibitory peptides significantly enhanced the efficacy of TNFalpha-induced killing. Notably, an inverse correlation is shown to exist between neutrophil APN activity and the sensitivity of donor cells to TNFalpha-induced apoptosis. Inhibition of cell surface APN appears to interfere with the shedding of TNFRI, and as a consequence results in augmented TNFalpha-induced apoptosis, cell polarization, and TNFalpha-primed, formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst. Of note, actinonin and bestatin had no effect on TNFRII expression under resting or TNFalpha-stimulated conditions and did not alter CXCRI or CXCRII expression. These data suggest significant variation in the activity of APN/CD13 on the cell surface of neutrophils in normal individuals and reveal a novel mechanism whereby APN/CD13 regulates TNFalpha-induced apoptosis via inhibition of TNFRI shedding. This has therapeutic relevance for driving neutrophil apoptosis in vivo.

C-Reactive Protein Products Generated by Neutrophil Elastase Promote Neutrophil Apoptosis

C-reactive protein (CRP), a prototypical acute phase protein, is increased as much as 1000-fold during acute inflammation, suggesting its biological role in that process. CRP can be modified at sites of inflammation where proteases have been released by neutrophils migrating to tissues. In this study, we investigated whether native CRP and neutrophil elastase-digested products of CRP modulate the rate of neutrophil apoptosis. Neutrophils were isolated from venous blood of healthy volunteers and incubated with either native CRP (1, 10, or 30 microg/mL) or CRP digested with neutrophil elastase (1 U/mL). After 6 and 24 h of incubation, neutrophils were harvested and examined for apoptosis under light microscopy. We found that CRP degradation products generated by neutrophil elastase, but not native CRP, promoted neutrophil apoptosis and cell death. The rate of apoptosis was not affected by the addition of elastase that was not incubated with CRP. These results suggest that CRP degradation products generated by neutrophil elastase promote neutrophil apoptosis. Cleavage of CRP by neutrophil elastase may offer protection from inflammatory injury.

The Involvement of the Apoptosis-Modulating Proteins ERK 1/2, Bcl-x L and Bax in the Resolution of Acute Inflammation in Vivo

Inflammatory cell recruitment, activation, and apoptosis are highly regulated processes involving several checkpoints controlling the resolution of inflammation. We investigated the role of the mitogen-activated protein kinase (MAPK) signaling pathway (ie, ERK1/2) and apoptosis-regulating Bcl-2 family members (ie, Bcl-x(L) and Bax) in the resolution of a rat carrageenan-induced pleurisy model. The specific ERK1/2 inhibitor PD98059 enhanced the resolution of inflammation, whereas the MEK1/2 inhibitor U0126 had no effect and the flavonoid apigenin, a nonspecific inhibitor of ERK1/2 and COX-2, augmented inflammation. Specifically, PD98059 significantly decreased the total number of macrophages and neutrophils in the pleural cavity, mainly by increasing the rate of neutrophil apoptosis, as measured by Annexin V labeling and morphological analysis. Conversely, a specific inhibitor of proapoptotic Bax (V5) increased inflammation, indicating that by preventing apoptosis in vivo, resolution of inflammation is delayed. This was associated with a decrease in neutrophil apoptosis and an increase in macrophage and neutrophil numbers perpetuating the inflammatory response. In conclusion, this study shows that ERK1/2, Bax, and Bcl-x(L) play important functional roles in the resolution phase of the acute inflammatory response in vivo by influencing apoptosis. Importantly, these data may provide novel therapeutic targets for the treatment of inflammatory diseases.

The effect of N-acetylcysteine on the rate of neutrophil apoptosis among anaesthetists

The effect of N-acetylcysteine on the rate of neutrophil apoptosis among anaesthetists

Granulocyte Macrophage Colony-stimulating Factor Signaling and Proteasome Inhibition Delay Neutrophil Apoptosis by Increasing the Stability of Mcl-1

Human neutrophils normally have a very short half-life and die by apoptosis. Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) can delay this apoptosis via increases in the cellular levels of Mcl-1, an anti-apoptotic protein of the Bcl-2 family with a rapid turnover rate. Here we have shown that inhibition of the proteasome (a) decreases the rate of Mcl-1 turnover within neutrophils and (b) significantly delays apoptosis. This led us to determine whether GM-CSF could enhance neutrophil survival by altering the rate of Mcl-1 turnover. Addition of GM-CSF to neutrophils enhanced Mcl-1 stability and delayed apoptosis by signaling pathways requiring PI3K/Akt and p44/42 Erk/Mek, because inhibitors of these pathways completely abrogated the GM-CSF-mediated effect on both Mcl-1 stability and apoptosis delay. Conversely, induction of Mcl-1 hyperphosphorylation by the phosphatase inhibitor, okadaic acid, significantly accelerated both Mcl-1 turnover and apoptosis. Neither the calpain inhibitor, carbobenzoxy-valinyl-phenylalaninal, nor the pan caspase inhibitor, benzyloxycarbonyl-VAD-fluoromethylketone, had any effect on Mcl-1 stability under these conditions. These observations indicate that profound changes in the rate of neutrophil apoptosis following cytokine signaling occur via dynamic changes in the rate of Mcl-1 turnover via the proteasome.

Regulation of granulocyte apoptosis by NF-kappaB.

Granulocyte apoptosis is a crucial part of the successful resolution of inflammation. In vitro results show that activation of NF-kappaB (nuclear factor kappaB) in granulocytes is a survival mechanism. NF-kappaB inhibitors increase the rate of constitutive apoptosis in neutrophils and eosinophils and cause these cells to respond to the pro-apoptotic effects of TNF-alpha (tumour necrosis factor-alpha). Results from both in vivo and in vitro experiments suggest that there are at least two important waves of NF-kappaB activation in inflammatory loci, which increase the expression of COX-2 (cyclooxygenase-2), itself an NF-kappaB controlled gene. The first wave causes the production of inflammatory mediators such as PGE2 (prostaglandin E2), allowing the establishment of inflammation. The second wave causes the synthesis of PGD2 and its metabolites that induce granulocyte apoptosis by inhibiting NF-kappaB activation. These metabolites may therefore be important physiological mediators controlling the resolution of inflammation. Although NF-kappaB is an important target for anti-inflammatory therapy, the timing of inhibition in vivo may be crucial, to ensure that production of PGD2 and its sequential metabolites can occur.

ASPIRIN PRESERVES NEUTROPHIL APOPTOSIS AFTER CARDIOPULMONARY BYPASS

The aim of this study was to test the hypothesis that ongoing aspirin therapy preserves neutrophil apoptosis after cardiac surgery with cardiopulmonary bypass (CPB) by a cyclooxygenase mechanism. Twenty patients undergoing coronary revascularization with CPB were enrolled in a prospective cohort study. Patients who had continued taking 300 mg of aspirin until the day before surgery (n = 10) were compared with 10 patients not taking aspirin or who had discontinued it more than 5 days before surgery. Neutrophils were isolated from arterial blood before and 6 h after surgery and apoptosis was measured after 24 h in culture using flow cytometry. Serum was collected and assessed for IL-6, IL-8 and PGE2 by enzyme-linked immunoabsorbant assay. Patients were followed for clinical indices of sepsis for 7 days postoperatively. Spontaneous rates of neutrophil apoptosis were significantly reduced in postoperative compared with preoperative samples. There was no difference between aspirin and control preoperative neutrophil apoptosis rates (23.0% +/- 11.3% vs. 23.0% +/- 20.7%, P = 0.99). Postoperative neutrophil apoptosis was delayed in control patients (3.6% +/- 1.2% apoptosis), but this was significantly (P = 0.045) reversed in the aspirin-treated group (7.2% +/- 5.1% apoptosis). There were lower postoperative PGE2 levels in the aspirin group (136 +/- 69 pg/mL vs. 372 +/- 210 pg/mL, P = 0.04). There was no difference in clinical indices of sepsis. We conclude that the delay in postoperative neutrophil apoptosis is significantly preserved in patients taking 300 mg of aspirin on the day before surgery. This was associated with greater inhibition of PGE2, consistent with the hypothesis that aspirin exerts its effect on apoptosis after CPB via a cyclooxygenase-mediated mechanism.

Elevated serum granulocyte colony-stimulating factor levels in patients with active phase of sweet syndrome and patients with active behcet disease: implication in neutrophil apoptosis dysfunction.

Sweet syndrome (SS), an acute inflammatory disease, has clinical and laboratory features similar to those of Behçet disease (BD). Serum levels of granulocyte colony-stimulating factor (G-CSF) are elevated in patients with SS, and exogenous administration of G-CSF has repeatedly been implicated in the causation of SS. Granulocyte colony-stimulating factor is a hematopoietic growth factor that regulates the production and differentiation of neutrophils. To clarify the role of elevated serum G-CSF levels in patients with active SS and active BD compared with those with inactive SS or BD and healthy controls. To then analyze neutrophil apoptosis in the active state of SS and BD; and to also investigate the influence of autologous serum on neutrophil apoptosis. Serum G-CSF was examined in 5 patients with active SS, 7 with inactive SS, 7 with active BD, 9 with inactive BD, and 5 healthy controls by means of an enzyme immunoassay kit. We measured apoptotic cells in the neutrophil fraction of peripheral blood collections in patients with active diseases and controls by means of flow cytometry. Serum G-CSF level was significantly higher in patients with active SS than in those with inactive SS. The difference in serum G-CSF levels among patients with active and inactive BD was also significant. Serum G-CSF level was significantly higher in patients with active SS than in those with active BD. Neutrophil apoptosis was significantly higher in patients with active SS than healthy controls. This increased apoptosis rate was also seen in patients with active BD. The increased rate of neutrophil apoptosis was significantly suppressed when the neutrophils were cultured for 18 hours in the presence of autologous active SS serum. Similarly, neutrophil apoptosis was suppressed in the presence of autologous serum in patients with active BD, but not significantly so. These findings indicate that increased production of G-CSF in patients with SS and BD may play an important role in the manifestation of these disorders. Given the suppression of neutrophil apoptosis in the active state in the presence of the influence of autologous serum, which includes elevated G-CSF level, we propose that serum G-CSF plays a significant role in the suppression of neutrophil apoptosis. Furthermore, G-CSF-induced suppression of neutrophil apoptosis appears to be deeply involved in the pathogenesis of SS and BD.

Enhanced Neutrophil Apoptosis in Neutropenic Patients with Hepatosplenic Schistosomiasis: Evidence of Serum Fas Ligand

Enhanced neutrophil apoptosis has been reported in neutropenic hepatosplenic schistosomiasis. The shortening of neutrophil survival via apoptosis may explain the neutropenia that occur in these patients. However, the regulation of neutrophil apoptosis in hepatosplenic schistosomiasis has not been clearly defined. Neutrophils harvested from neutropenic patients with hepatosplenic (HS) schistosomiasis, (n=25), non-neutropenic patients with hepatointestinal (HI) schistosomiasis (n=10), and age-/gender-matched healthy control subjects (n=10) were incubated with autologous serum. Neutrophils apoptosis was quantified by flow cytometry through determination of propidium iodide nuclear staining and confirmed by DNA gel electrophoresis at 0 (i.e. fresh neutrophils), 4 and 24 h culture. Neutrophils from healthy subjects were also incubated with either 10% heterologous normal or neutropenic serum, with and without anti-Fas ligand antibody. Fas expression was assessed in fresh neutrophils using flow cytometry. Compared with normal healthy neutrophils, and HI neutrophils, neutropenic neutrophils demonstrated greater apoptosis in the presence of autologous serum (P<0.01, 0.05, respectively). Furthermore, compared with normal neutrophils exposed to heterologous normal serum, those exposed to heterologous neutropenic serum exhibited higher apoptosis rates ( P<0.01). Moreover, anti-Fas L antibody attenuated the neutropenic serum-induced neutrophil apoptosis in normal neutrophils. Fas expression was significantly higher in the neutropenic group when compared to both HI and normal healthy controls (P<0.05). In addition, Fas expression by neutrophils was paralleled by high neutrophil apoptosis. On the other hand, neutrophil apoptosis was not correlated to the size of spleen in neutropenic group.In conclusion, the rate of neutrophil apoptosis is accelerated in patients with neutropenic hepatosplenic schistosomiasisis. These findings suggest that the enhanced neutrophil apoptosis that occurs in neutropenic HS patients is triggered by a serum factor, which is mostly a Fas ligand.

Accelerated neutrophil apoptosis in neutropenic patients with hepatosplenic schistosomiasis is induced by serum Fas ligand.

Neutropenia in patients with hepatosplenic (HS) schistosomiasis may stem from enhanced neutrophil apoptosis. However, the molecular mechanism of neutrophil apoptosis has not been clearly defined. Neutrophils harvested from neutropenic patients with HS schistosomiasis (n = 25), non-neutropenic patients with hepatointestinal (HI) schistosomiasis (n = 10), and age- and sex-matched healthy control subjects (n = 10) were examined for the degree of apoptosis after incubation with autologous sera. Neutrophil apoptosis was quantified by flow cytometry through determination of propidium iodide nuclear staining and confirmed by DNA gel electrophoresis at 0 time (fresh neutrophil), 4 and 24 h culture. Neutrophils from healthy subjects were also incubated with either 10% heterologous normal or neutropenic serum, with and without anti-Fas ligand antibody. Serum Fas ligand levels were assessed in sera of patient groups and healthy controls by ELISA. Compared with normal controls and HI, HS group demonstrated greater neutrophil apoptosis in the presence of autologous serum (P < 0.01, < 0.05, respectively). Furthermore, compared with normal neutrophils exposed to heterologous normal serum, those exposed to heterologous neutropenic serum exhibited higher apoptosis rates (P < 0.01). The apoptotic effect of neutropenic sera is attenuated by anti-Fas ligand. Fas expression was significantly higher in HS group as compared to both HI and normal healthy controls (P < 0.05). Serum Fas ligand levels were significantly higher among HS group as compared to both HI and control groups (P < 0.01 for both). Neutrophil apoptosis was not correlated to the size of spleen in HS group. In conclusion, the rate of neutrophil apoptosis is accelerated in neutropenic HS schistosomiasis. These findings suggest that enhanced neutrophil apoptosis demonstrated in HS patients is triggered by soluble Fas ligand, which is mostly derived from spleen.

Ca2+ influx shutdown during neutrophil apoptosis: importance and possible mechanism.

Neutrophils are essential components of the natural immune system and form the first line of defence against bacterial and fungal infections. They are terminally differentiated cells and have half-lives in the circulation of only 8–20 hr. Their nuclei are invaginated to form a number of defined lobes with thin connecting filaments.1 The DNA is condensed within these nuclear lobes and is randomly distributed into the separate lobes2,3 and probably cannot be transcribed. These cells also have little endoplasmic reticulum (ER), but can produce some protein from existing mRNA.4 They have few mitochondria and the cells generate ATP glycolytically. The long-term survival of these cells is thus insecure, and ultimately limited by the extent of their glycogen stores. One route towards death would thus occur as ATP levels diminished and ion pumps, especially plasma membrane Ca2+-ATPase, became progressively ineffective until cell death proceeds by necrosis. However, it may result in the release of hydrolases and proteases either by necrotic lysis or by Ca2+-driven exocytosis. Moreover, extracellular superoxide generation may also be triggered by Ca2+ influx.5–8 Thus, potentially disastrous extensive damage to the tissues may occur. It thus seems unlikely that the elimination of the millions of neutrophils that die physiologically would be by the necrotic route. Furthermore, neutrophil necrosis is unlikely to be the route for neutrophil clearance in inflammation. Instead, at the resolution of inflammation the neutrophil activity is reduced by an increase in the rate of neutrophil apoptosis. While our understanding of this physiological process of apoptosis as a cell biological phenomenon has increased over the last decade, the unusual role of apoptosis for neutrophils and its consequences are only now being understood. For example, in many cells, apoptosis is characterized by nuclear condensation and invagination, a morphological event that occurs in neutrophils as they mature. In some respects then the mature neutrophil has already embarked on the route towards apoptosis. This is also evident as neutrophils undergo apoptosis spontaneously, without the need for external stimuli. In fact, external stimuli may modify the rate of this constitutive apoptosis, either by accelerating or delaying cell death, but cannot prevent it.

Benzodiazepines inhibit the rate of neutrophil apoptosis.

Benzodiazepines, which are commonly administered perioperatively, can depress immune function. Neutrophil apoptosis plays a central role in the regulation of inflammation. This is particularly important during and after surgery. To examine the effects of benzodiazepines (midazolam and diazepam) on neutrophil apoptosis. Venous blood samples were withdrawn from patients scheduled to undergo elective surgery, (a) immediately prior to, and 10 minutes after administration of midazolam 0.2 mg/kg intravenously (n=11) and (b) immediately prior to, and 60 minutes after administration of diazepam 10 mg p.o. (n=10). Neutrophil apoptosis was measured by Annexin V-FITC after 1 and 12 hours in culture. The percentage of apoptotic cells was significantly less after midazolam at 12% (11.9) hours in culture compared to pre-midazolam 29.7% (13.3) (p<0.05). After diazepam, the rates of neutrophil apoptosis were also significantly less after 12 hours in culture (p<0.05). Administration of benzodiazepines in clinically relevant doses inhibits neutrophil apoptosis. In the perioperative period, this may influence the inflammatory response to surgery.

Nitric oxide: a key regulator of myeloid inflammatory cell apoptosis.

Apoptosis of inflammatory cells is a critical event in the resolution of inflammation, as failure to undergo this form of cell death leads to increased tissue damage and exacerbation of the inflammatory response. Many factors are able to influence the rate of apoptosis in neutrophils, eosinophils, monocytes and macrophages. Among these is the signalling molecule nitric oxide (NO), which possesses both anti- and proapoptotic properties, depending on the concentration and flux of NO, and also the source from which NO is derived. This review summarises the differential effects of NO on inflammatory cell apoptosis and outlines potential mechanisms that have been proposed to explain such actions.

The mitochondrial network of human neutrophils: role in chemotaxis, phagocytosis, respiratory burst activation, and commitment to apoptosis.

It is commonly assumed that human neutrophils possess few, if any, functional mitochondria and that they do not depend on these organelles for cell function. We have used the fluorescent mitochondrial indicators, JC-1, MitoTracker Red, and dihydrorhodamine 123 to show that live neutrophils possess a complex mitochondrial network that extends through the cytoplasm. The membrane potential of these mitochondria was rapidly (within 2 min) disrupted by the addition of FCCP (IC(50) = 20 nM), but not by the Fo-ATPase inhibitor, oligomycin (at up to 7 microg/ml). However, inhibition of mitochondrial function with both agents resulted in cell shape changes. Neither activation of the respiratory burst nor phagocytosis of either latex particles or serum-opsonized Staphylococcus aureus was affected by the addition of FCCP or oligomycin. However, FCCP inhibited chemotaxis at concentrations that paralleled disruption of mitochondrial membrane potential. Furthermore, prolonged (2-h) incubation with oligomycin resulted in an impaired ability to activate a respiratory burst and also inhibited chemotaxis. These observations indicate that intact mitochondrial function is required to sustain some neutrophil functions, but not for the rapid initiation of the respiratory burst or phagocytosis. Loss of mitochondrial membrane potential was a very early marker for commitment of neutrophils into apoptosis and preceded the appearance of phosphatidylserine on the cell surface. However, inhibition of mitochondrial function did not accelerate the rate of neutrophil apoptosis. These data shed important insights into the hitherto unrecognized importance of mitochondria in the function of neutrophils during infection and inflammation.

Role of PI3-kinase–dependent Bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival

Phosphoinositide 3-kinase (PI3-kinase)-dependent phosphorylation of the proapoptotic Bcl-2 family member Bad has been proposed as an important regulator of apoptotic cell death. To understand the importance of this pathway in nontransformed hematopoietic cells, we have examined the effect of survival cytokines on PI3-kinase activity and Bad expression and phosphorylation status in human neutrophils. Granulocyte macrophage-colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) both reduced the rate of apoptosis in neutrophils cultured in vitro for 20 hours. Coincubation with the PI3-kinase inhibitor LY294002, which in parallel experiments abolished GM-CSF-primed, fMLP-stimulated superoxide anion production and GM-CSF-stimulated PtdIns(3,4,5)P(3) accumulation, inhibited the GM-CSF and TNF-alpha survival effect. In contrast, the MAP kinase kinase (MEK1/2) inhibitor PD98059 and the protein kinase A inhibitor H-89 had only a marginal effect on GM-CSF-mediated neutrophil survival. GM-CSF substantially increased Bad phosphorylation at Ser112 and Ser136 and increased the cytosolic accumulation of Bad. GM-CSF also regulated Bad at a transcription level with a marked decrease in mRNA levels at 4 hours. TNF-alpha caused a biphasic effect on the rate of morphologic apoptosis, which corresponded to an early increase, and a late inhibition, of Bad mRNA levels. LY294002 inhibited GM-CSF- and TNF-alpha-mediated changes in Bad phosphorylation and mRNA levels. These data suggest that the survival effect of GM-CSF and TNF-alpha in neutrophils is caused by a PI3-kinase-dependent phosphorylation and cytosolic translocation of Bad, together with an inhibition of Bad mRNA levels. This has important implications for the regulation of neutrophil apoptosis in vivo.