Apoptosis Of Polymorphonuclear Neutrophils Research Articles

The mechanism of Xuebijing injection in preventing and treating lung injury induced by cardiopulmonary bypass by regulating the apoptosis of alveolar polymorphonuclear neutrophil

To investigate the protective effect of Xuebijing injection on acute lung injury (ALI) associated with cardiopulmonary bypass (CPB) by regulating the apoptosis of polymorphonuclear neutrophils (PMN). Thirty male Sprague-Dawley (SD) rats were randomly divided into sham operation group (Sham group), CPB model group (CPB group) and Xuebijing pretreatment group (XBJ group) according to the random number table method, with 10 rats in each group. Rats in the CPB group and XBJ group undergoing CPB procedures for 60 minutes. Rats in the Sham group did not undergo CPB. Rats in the XBJ group received intraperitoneal injection of 4 mL/kg Xuebijing injection 2 hours before CPB. Rats in the Sham group and CPB group were injected with an equal amount of normal saline. 4 hours after CPB, arterial blood was collected for blood gas analysis to calculate respiratory index (RI), and lung tissue of rats was collected for determination of lung index (LI) and pulmonary water containing rate. PMN in bronchoalveolar lavage fluid (BALF) were collected and the activity of caspase-3 was detected. The apoptosis rate was detected by flow cytometry. The expressions of microRNA-142-3p (miR-142-3p) and FoxO1 mRNA were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The protein expression of FoxO1 was detected by Western blotting. In addition, HL-60 cells were divided into control oligonucleotide transfection group, miR-142-3p mimics transfection group, and miR-142-3p inhibitor transfection group. After 48 hours of transfection, the activity of miR-142-3p binding to FoxO1 was detected using dual luciferase reporter genes. Compared with Sham group, RI, LI and pulmonary water containing rate were significantly increased in CPB group. The caspase-3 activity and apoptosis rate of PMN obtained from BALF were significantly decreased, the expression of miR-142-3p was decreased, and the expression of FoxO1 protein was increased. However, compared with CPB group, RI, LI and pulmonary water containing rate were significantly decreased in XBJ group [RI: 0.281±0.066 vs. 0.379±0.071, LI: 4.50±0.26 vs. 5.71±0.42, pulmonary water containing rate: (80.31±32.50)% vs. (84.59±3.41)%, all P < 0.01]. The caspase-3 activity and apoptosis rate of PMN obtained from BALF were significantly increased [caspase-3 activity: 0.350±0.021 vs. 0.210±0.014, apoptosis rate: (15.490±1.382)% vs. (8.700±0.701)%, both P < 0.01], the expression of miR-142-3p was significantly up-regulated (2-ΔΔCt: 2.61±0.17 vs. 0.62±0.05, P < 0.01), and the protein expression of FoxO1 was decreased [FoxO1/GAPDH (relative expression level): 0.81±0.04 vs. 1.22±0.06, P < 0.01]. However, there was no statistically significant difference in FoxO1 mRNA expression among the three groups. The bioinformatics analysis results showed that miR-142-3p can bind to the FoxO1 3'untranslated region (3'UTR). In HL-60 cells, compared with control oligonucleotide transfection group, the transfection of miR-142-3p mimics could reduce the expression of FoxO1 protein [FoxO1/GAPDH (relative expression level): 0.48±0.06 vs. 1.00±0.05, P < 0.01], however, the transfection of miR-142-3p inhibitor increased the expression of FoxO1 protein [FoxO1/GAPDH (relative expression level): 1.37±0.21 vs. 1.00±0.05, P < 0.05]. But, transfection with miR-142-3p mimics or inhibitor had no effect on FoxO1 mRNA expression. The luciferase reporter gene showed that miR-142-3p could bind to the FoxO1 3'UTR to inhibit FoxO1 expression. Xuebijing injection may promote the apoptosis of pulmonary alveolar PMN through the miR-142-3p/FoxO1 axis, and play a role in the prevention and treatment of CPB-induced ALI.

Long non-coding RNA X-inactive specific transcript silencing ameliorates primary graft dysfunction following lung transplantation through microRNA-21-dependent mechanism.

BackgroundPrimary graft dysfunction (PGD) is a known acute lung injury (ALI) and a major cause of fatality post-lung transplantation. Though some long non-coding RNAs (lncRNAs) have been studied in ALI through regulation of microRNAs (miRNAs), their effects on PGD remain undefined. The present study aims to explore the underlying mechanism of lncRNA X-inactive specific transcript (XIST) in PGD after lung transplantation.MethodsInitially, the expression of miR-21, IL-12A and XIST was determined by RT-qPCR and western blot analysis. The dual luciferase reporter assay, RNA pull-down and RIP assay were performed to identify the targeting relationship between miR-21 and IL-12A and the binding relationship between miR-21 and XIST. Loss- and gain-of-function investigations were conducted in rats treated with prolonged cold ischemia and polymorphonuclear neutrophils (PMNs).FindingsmiR-21 was decreased, whilst XIST and IL-12A were increased in the bronchoalveolar lavage fluid of PGD patients after lung transplantation. Enhanced miR-21 expression in rats and PMNs resulted in downregulated expression of pro-inflammatory factors and chemokines, and enhanced the apoptosis of PMNs. XIST was found to upregulate IL-12A expression in a miR-21-dependent manner. Additionally, XIST silencing enhanced the apoptosis of PMNs and inhibited the neutrophil extracellular trap (NET) formation through upregulation of miR-21 but downregulation of IL-12A in vivo.InterpretationIn summary, lncRNA XIST upregulates IL-12A by binding to miR-21, thereby inducing NET formation and accelerating PGD after lung transplantation. This suggests that inhibition of XIST and NET may be beneficial for the treatment of PGD.

Emodin alleviates severe acute pancreatitis-associated acute lung injury by decreasing pre-B-cell colony-enhancing factor expression and promoting polymorphonuclear neutrophil apoptosis.

The present study aimed to evaluate the protective effects of emodin on severe acute pancreatitis (SAP)-associated acute lung injury (ALI), and investigated the possible mechanism involved. SAP was induced in Sprague-Dawley rats by retrograde infusion of 5% sodium taurocholate (1 ml/kg), after which, rats were divided into various groups and were administered emodin, FK866 [a competitive inhibitor of pre-B-cell colony-enhancing factor (PBEF)] or dexamethasone (DEX). DEX was used as a positive control. Subsequently, PBEF expression was detected in polymorphonuclear neutrophils (PMNs) isolated from rat peripheral blood by reverse transcription-quantitative polymerase chain reaction and western blotting. In addition, histological alterations, apoptosis in lung/pancreatic tissues, apoptosis of peripheral blood PMNs and alterations in the expression of apoptosis-associated proteins were examined by hematoxylin and eosin staining, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay, Annexin V/propidium iodide (PI) assay and western blotting, respectively. Serum amylase activity and wet/dry (W/D) weight ratios were also measured. An in vitro study was also conducted, in which PMNs were obtained from normal Sprague-Dawley rats and were incubated with emodin, FK866 or DEX in the presence of lipopolysaccharide (LPS). Apoptosis of PMNs and the expression levels of apoptosis-associated proteins were examined in cultured PMNs in vitro by Annexin V/PI assay and western blotting, respectively. The results demonstrated that emodin, FK866 and DEX significantly downregulated PBEF expression in peripheral blood PMNs. In addition, emodin, FK866 and DEX reduced serum amylase activity, decreased lung and pancreas W/D weight ratios, alleviated lung and pancreatic injuries, and promoted PMN apoptosis by regulating the expression of apoptosis-associated proteins: Fas, Fas ligand, B-cell lymphoma (Bcl)-2-associated X protein, cleaved caspase-3 and Bcl-extra-large. In addition, the in vitro study demonstrated that emodin, FK866 and DEX significantly reversed the LPS-induced decrease of apoptosis in PMNs by regulating the expression of apoptosis-associated proteins. In conclusion, the present study demonstrated that emodin may protect against SAP-associated ALI by decreasing PBEF expression, and promoting PMN apoptosis via the mitochondrial and death receptor apoptotic pathways.

Unconventional apoptosis of polymorphonuclear neutrophils (PMN): staurosporine delays exposure of phosphatidylserine and prevents phagocytosis by MΦ-2 macrophages of PMN

Apoptosis of polymorphonuclear neutrophils (PMN) and subsequent 'silent' removal represents an important check-point for the resolution of inflammation. Failure in PMN clearance resulting in secondary necrosis-driven tissue damage has been implicated in conditions of chronic inflammation and autoimmunity. Apoptotic PMN undergo profound biophysical changes that warrant their efficient recognition and uptake by phagocytes before fading to secondary necrosis. In this study, we demonstrate that staurosporine (STS), a non-selective but potent inhibitor of cyclin-dependent kinase and protein kinase C, exerts a drastic impact on PMN apoptosis. PMN treated with STS underwent an unconventional form of cell death characterized by a delayed exposure of aminophospholipids, including phosphatidylserine (PS) and phosphatidylethanolamine and an increased exposure of neo-glycans. STS caused an impaired cellular fragmentation and accelerated DNA fragmentation. Phagocytosis of STS-treated PMN lacking PS on their surfaces was decreased significantly, which highlights the importance of PS for the clearance of apoptotic PMN. Specific opsonization with immune complexes completely restored phagocytosis of STS-treated PMN, demonstrating the efficiency of back-up clearance pathways in the absence of PS exposure.

Prehospital Hypertonic Saline Resuscitation Attenuates the Activation and Promotes Apoptosis of Neutrophils in Patients With Severe Traumatic Brain Injury

Activation of polymorphonuclear neutrophils (PMNs) is thought to contribute to traumatic brain injury (TBI). Since hypertonic fluids can inhibit PMN activation, we studied whether hypertonic fluid resuscitation can reduce excessive PMN activation in TBI patients. Trauma patients with severe TBI were resuscitated with 250 mL of either 7.5% hypertonic saline (HS; n = 22), HS + 6% dextran-70 (HSD; n = 22), or 0.9% normal saline (NS; n = 39), and blood samples were collected on hospital admission and 12 and 24 h after resuscitation. Polymorphonuclear neutrophil activation (CD11b, CD62L, CD64) and degranulation (CD63, CD66b, CD35) markers and oxidative-burst activity, as well as spontaneous PMN apoptosis were measured by flow cytometry. Relative to healthy controls, TBI patients showed increased PMN activation and decreased apoptosis of PMNs. In the HS group, but not in the HSD group, markers of PMN adhesion (CD11b, CD64) and degranulation (CD35, CD66b) were significantly lower than those in the NS group. These effects were particularly pronounced 12 h after resuscitation. Treatment with HS and HSD inhibited PMN oxidative burst responses compared with NS-treated patients. Hypertonic saline alone partially restored delayed PMN apoptosis. Despite these differences, the groups did not differ in clinical outcome parameters such as mortality and Extended Glasgow Outcome Scale. This study demonstrates that prehospital resuscitation with HS can partially restore normal PMN activity and the apoptotic behavior of PMNs, whereas resuscitation with HSD was largely ineffective. Although the results are intriguing, additional research will be required to translate these effects of HS into treatment strategies that improve clinical outcome in TBI patients.

Clinical Isolates ofMycobacterium tuberculosisDiffer in Their Ability to Induce Respiratory Burst and Apoptosis in Neutrophils as a Possible Mechanism of Immune Escape

Tuberculosis pathogenesis was earlier thought to be mainly related to the host but now it appears to be clear that bacterial factors are also involved. Genetic variability of Mycobacterium tuberculosis (Mtb) could be slight but it may lead to sharp phenotypic differences. We have previously reported that nonopsonized Mtb H37Rv induce apoptosis of polymorphonuclear neutrophils (PMNs) by a mechanism that involves the p38 pathway. Here we evaluated the capability to induce PMN apoptosis of two prevalent Mtb lineages in Argentina, the Latin America and Mediterranean (LAM), and Haarlem, using the H37Rv as a reference strain. Results showed that LAM strains strongly induced apoptosis of PMN which correlated with the induction of reactive oxygen species (ROS) production and p38 activation. Interestingly, the highly prosperous multidrug-resistant M strain, belonging to the Haarlem lineage, lacked the ability to activate and to induce PMN apoptosis as a consequence of (1) a weak ROS production and (2) the contribution of antiapoptotic mechanisms mediated at least by ERK. Although with less skill, M is able to enter the PMN so that phenotypic differences could lead PMN to be a reservoir allowing some pathogens to prevail and persist over other strains in the community.

Immunostimulatory properties of the bacterial extract OM-89 in vitro and in vivo

The therapeutic agent OM-89 (Uro-Vaxom) contains lyophilized immunostimulating fractions from 18 Escherichia coil strains. It has been shown to provide protection against recurrent urinary tract infections in humans and against bacterial infections in mice. Here the immunostimulatory properties of OM-89 were investigated by in vitro and in vivo assays. In vitro the activation of murine spleen cells by the AlamarBlue assay was determined. OM-89 was effective in stimulating the metabolism of spleen cells within a concentration range of 0.625-2.5 mg/ml. The activation of murine bone marrow-derived macrophages by OM-89 was shown by the induction of NO production; OM-89 was a most effective stimulant at concentrations around 6 mg/ml. In the human system, the effect of OM-89 was tested in vitro:metabolic activity of peripheral blood lymphocytes (PBL) was stimulated starting at concentrations of approx. 250 microg/ml, and the spontaneous apoptosis of polymorphonuclear neutrophils (PMN) was reduced starting at OM-89 concentrations of approx. 100 microg/ml. Finally, in a mouse model, the in vivo protection of mice against infection with Salmonella typhimurium after the oral administration of OM-89 was tested (100 mg in a volume of 0.5 ml once a day for 10 consecutive days). The extract proved to be effective: 90% of the OM-89-treated animals survived compared to 58% of the untreated control group.

English

&nbsp; Previous studies have shown that delayed apoptotic polymorphonuclear neutrophils (PMNs) might play an important role in the development of multiple organ dysfunction syndromes (MODS). Somatostatin (SST) may improve the histopathological lesions as well as functions of vital organs in rats with MODS. This study was to investigate the association of SST levels in plasma or jejunum tissue and PMN life in macaques following intestinal ischemia reperfusion. SST levels in macaque plasma or jejunum tissue were significantly decreased after intestinal ischemia reperfusion (IR) injury. The apoptotic rate of PMNs was obviously reduced (15.4&plusmn;1.41 to 3.5&plusmn;0.53%,&nbsp;P&lt;0.05). However, the number of peritoneal&nbsp;apoptotic&nbsp;macrophages&nbsp;was clearly increased. The apoptotic rate of PMNs was significantly increased (20.0&plusmn;2.24 to 50.2&plusmn;1.81%,&nbsp;P&lt;0.01)&nbsp;after incubated with&nbsp;SST&nbsp;in vitro.&nbsp;Specific&nbsp;DNA ladder bends from PMNs were visualized with agarose electrophoresis.&nbsp;Specific bindings between&nbsp;PMNs and the antibodies for SSTR1 (547.9&plusmn;19.98 RU/ml) or SSTR2 (27.6&plusmn;20.56 RU/ml) were detected. The results showed physiological apoptosis of PMNs in macaques could be induced by SST through SSTRs on PMNs. The decreased SST levels in macaque plasma or jejunum tissue following intestinal ischemia-reperfusion might extend PMN life and promote the occurrence of MODS. &nbsp; Key words:&nbsp;Polymorphonuclear neutrophils (PMNs), macrophage, apoptosis, somatostatin,&nbsp;macaque,&nbsp;multiple organ dysfunction syndromes (MODS).

Delayed neutrophil apoptosis in patients with unstable angina: relation to C-reactive protein and recurrence of instability

To investigate spontaneous polymorphonuclear neutrophils (PMNs) apoptosis in unstable angina (UA) and its association with recurrence of instability. We compared PMNs apoptotic rate at 4 and 24 h in patients with UA, stable angina (SA), and controls (H) with two different protocols by flow cytometry. We measured apoptotic rate of isolated PMNs (Protocol 1) in 30 UA patients, 13 SA patients, and 34 H; and apoptosis of PMNs in whole blood culture (Protocol 2) in further 10 UA patients, 7 SA patients, and 6 H. Serum high-sensitivity C-reactive protein was also measured. Polymorphonuclear neutrophils of UA patients showed a decreased apoptotic rate compared with SA patients and H at 4 h in Protocol 1 (both P < 0.01), and at 24 h in Protocol 2 (P < 0.05 and <0.01, respectively). In overall population, a negative correlation was found between apoptotic rate at 4 h and high-sensitivity C-reactive protein levels (P < 0.01). Six among 40 patients with UA had early recurrence of symptoms and their apoptotic rate was significantly reduced compared with UA patients without recurrence of symptoms (P = 0.024). Our study demonstrates delayed PMN apoptosis in UA. This alteration might be involved in the persistence of inflammatory activation and affects recurrence of instability.

The expressions of intrinsic and extrinsic apoptotic pathway proteins in neutrophils of oral cavity cancer patients: a preliminary study

The biological availability and activity of polymorphonuclear neutrophils (PMNs) are regulated by their short life span, which can be additionally shortened by a malignant process. The signaling pathways leading to apoptotic PMN death are classified in two categories: the intrinsic and the extrinsic. In the present study the expressions of proteins participating in the extrinsic apoptotic pathway (DR5, FADD, caspase-8 activity) and the intensity of apoptosis of PMNs from patients with cancer of the oral cavity were examined. The expression of proteins participating in the intrinsic pathway (Bax and Mcl-1) were also examined in these cells. The results can be helpful in explaining the reasons for the decreased activity of these cells in oral cavity cancer patients. The examinations were carried out in patients with squamous cell carcinoma of the oral cavity before and after treatment. The expressions of all the proteins were measured in neutrophils and, for comparison, in autologous peripheral blood mononuclear cells (PBMCs). Western blot analysis was used to assay the expressions of DR5, FADD, Bax, and Mcl-1 in cell lysates. The apoptosis level was determined by flow cytometry and caspase-8 activity by colorimetric assay. A lack of changes in DR5 expression associated with increased FADD protein expression and caspase-8 activity accompanied the accelerated apoptosis rates in the PMNs of the patients before treatment. Decreased expression of anti-apoptotic Mcl-1 protein was associated with an unchanged expression of pro-apoptotic Bax protein. There were no such changes in the patients PBMCs. Increased expression of Mcl-1 in the PMNs of the patients following surgical treatment was found. The acceleration of the apoptosis of PMNs of oral cavity cancer patients before treatment is dependent on both the intrinsic and extrinsic pathways.

Delayed apoptosis of circulating neutrophils in Kawasaki disease.

Circulating polymorphonuclear neutrophils (PMNs) are known to increase in number and are functionally activated in the acute phase of Kawasaki disease (KD). In the present study, we investigated whether the apoptosis of PMNs is deregulated in KD. When the isolated PMNs were cultured in vitro, the proportions of spontaneous apoptotic PMNs (annexin V+ cells and cells with fragmented DNA) were found to be significantly lower (P < 0.01) in the patients with KD (n = 25) than in the patients with a bacterial infection (n = 20) or a viral infection (n = 20), or in healthy children (n = 20). The proportion of circulating Fas-positive PMNs was also significantly lower (P < 0.01) in the acute KD patients than in the other groups. In the acute phase of KD, the proportion of spontaneous apoptotic PMNs showed a significant positive correlation (P < 0.01) with the proportions of circulating Fas-positive PMNs. Furthermore, the agonistic anti-Fas MoAb (CH-11) induced a significant increase in the proportion of apoptotic PMNs in the patients with a viral infection and healthy children, but not in either the patients with KD or the patients with a bacterial infection. In the intracellular expression of anti- and pro-apoptotic proteins, the A1/Bax ratio was significantly higher in acute KD than in the other groups. These findings indicate that PMN apoptosis is inhibited during the acute phase of KD and also suggest that both the resistance against the Fas-mediated death signal and the down-regulation of the mitochondrial apoptotic signalling pathway due to an altered balance of Bcl-2 protein expression are responsible for the delayed PMN apoptosis.

High Telomerase Activity in Neutrophils From Unstable Coronary Plaques

We evaluated telomerase activity in circulating polymorphonuclear neutrophils (PMN) and in PMN isolated from coronary atherosclerotic plaques by a novel approach. Delayed apoptosis of PMN have been demonstrated in unstable angina (UA). These cells have a finite lifespan with low telomerase activity, a polymerase that extends telomeres, structures essential for cell aging. Reactivation of telomerase has been associated with resistance to apoptosis. We studied 20 patients with UA and 6 patients with chronic stable angina (SA), undergoing a percutaneous coronary intervention. Circulating PMN were isolated from venous blood and PMN derived from coronary plaque were isolated from washing medium of angioplasty balloons. Telomerase activity was higher in coronary plaque PMN of UA patients than in coronary plaque PMN of SA patients (122.7, range 20.5 to 3,696; and 47.7, range 16 to 212.6, respectively, p = 0.001) and higher than in peripheral PMN of SA patients (122.7, range 20.5 to 3,696 vs. 59, range 16.5 to 132.5, p = 0.001). We found a statistically significant difference between venous and coronary plaque PMN telomerase activity in UA patients (z = -2.875; p = 0.004). Among UA patients, a shorter time interval from symptom onset to coronary PMN sampling was the only independent predictor of high telomerase activity in coronary plaque PMN (p < 0.001, R2 = 0.75). In UA patients, telomerase activity is high in coronary plaque PMN, while it is low in peripheral PMN. Telomerase reactivation in resident PMN resulting in a prolonged lifespan might play a key role in the early phases of instability.

Sevoflurane-induced oxidative stress and cellular injury in human peripheral polymorphonuclear neutrophils

Sevoflurane is an inhalation anesthetic used for general anesthesia. Several studies have demonstrated that reactive oxygen species (ROS) exist in cardioprotection when preconditioned with sevoflurane. Moreover, sevoflurane can also directly trigger the formation of peroxynitrite. Up to now, information pertinent to the effect of sevoflurane on cellular injuries in human polymorphonuclear neutrophils (PMN) is scant. In this study, we demonstrated that sevoflurane significantly increases intracellular H 2O 2 and/or peroxide, superoxide, and nitric oxide (NO) in PMN within 1 h treatment. Intensification of intracellular glutathione (GSH) depletion in PMN has been demonstrated with the presence of sevoflurane. Inhibition of sevoflurane-mediated intracellular H 2O 2 and/or peroxide in PMN by catalase, mannitol, dexamethasone, N-acetylcysteine (NAC) and trolox, but not superoxide dismutase (SOD) pretreatment, was observed. Among them, catalase has the best effect scavenging intracellular H 2O 2 and/or peroxide, suggesting that H 2O 2 is the major ROS during sevoflurane treatment. Two apoptotic critical factors—lowering of the mitochondrial transmembrane potential (Δ Ψ m) and activation of caspase 3/7—were significantly increased after 1 h of sevoflurane treatment. Apoptosis of PMN were determined by comet assay and flow cytometric analysis of annexin V-FITV protein binding to the cell surface. Exposure of PMN to sevoflurane markedly increased apoptosis in a dose-dependent manner. In summary, these results are important for demonstrating the oxidative stress and cellular injury on sevoflurane-treated human PMN.

Regulation of constitutive neutrophil apoptosis by the α,β-unsaturated aldehydes acrolein and 4-hydroxynonenal

Reactive alpha,beta-unsaturated aldehydes are major components of common environmental pollutants and are products of lipid oxidation. Although these aldehydes have been demonstrated to induce apoptotic cell death in various cell types, we recently observed that the alpha,beta-unsaturated aldehyde acrolein (ACR) can inhibit constitutive apoptosis of polymorphonuclear neutrophils and thus potentially contribute to chronic inflammation. The present study was designed to investigate the biochemical mechanisms by which two representative alpha,beta-unsaturated aldehydes, ACR and 4-hydroxynonenal (HNE), regulate neutrophil apoptosis. Whereas low concentrations of either aldehyde (<10 microM) mildly promoted apoptosis in neutrophils (reflected by increased phosphatidylserine exposure, caspase-3 activation, and mitochondrial cytochrome c release), higher concentrations prevented critical features of apoptosis (caspase-3 activation, phosphatidylserine exposure) and caused delayed neutrophil cell death with characteristics of necrosis/oncosis. Inhibition of caspase-3 activation by either aldehyde occurred despite increases in mitochondrial cytochrome c release and occurred in close association with depletion of cellular GSH and with cysteine modifications within caspase-3. However, procaspase-3 processing was also prevented, because of inhibited activation of caspases-9 and -8 under similar conditions, suggesting that ACR (and to a lesser extent HNE) can inhibit both intrinsic (mitochondria dependent) and extrinsic mechanisms of neutrophil apoptosis at initial stages. Collectively, our results indicate that alpha,beta-unsaturated aldehydes can inhibit constitutive neutrophil apoptosis by common mechanisms, involving changes in cellular GSH status resulting in reduced activation of initiator caspases as well as inactivation of caspase-3 by modification of its critical cysteine residue.

Perioperative treatment with human growth hormone down-regulates apoptosis and increases superoxide production in PMN from patients undergoing infrarenal abdominal aortic aneurysm repair

Background Recombinant human growth hormone (hGH) therapy has a beneficial effect on catabolism and wound healing after major surgery. Polymorphonuclear neutrophils (PMN) play an important role in this context. In a prospective, double-blind, randomized, placebo-controlled trial we studied the effect of perioperative hGH treatment on postoperative wound healing and on changes in superoxide generation and susceptibility to apoptosis of PMN in elderly patients undergoing elective abdominal aortic aneurysm repair. Methods Seven patients were treated with high-dose hGH (16 U/d) for nine days, seven patients with a placebo. IGF-I, neutrophil count, O 2-production induced by opsonized zymosan and apoptosis of PMN were measured and correlated with clinical outcome. Results Perioperative hGH treatment more than doubled the O 2 - production in PMN before and 24 h after surgery ( p < 0.01). The long-term capacity of PMN to generate O 2 in vitro was prolonged ( p < 0.001) in the hGH group. Spontaneous and Fas-inducible apoptosis was strongly down-regulated in PMN after surgery in all patients ( p < 0.01). hGH-treatment distinctly reduced apoptosis in PMN before and after surgery ( p < 0.01). Clinical outcome was similar in both groups. Conclusion Perioperative hGH treatment results in an enhanced O 2 - production in PMN and in a prolongation of the functional life span of these cells. This may improve immune function and help to overcome the postoperative anergic state of the immune system especially in elderly individuals.

Identification of caspase-10 in human neutrophils and its role in spontaneous apoptosis.

In the present study, we investigated the molecular mechanisms of spontaneous and tumor necrosis factor alpha (TNF-alpha)-mediated apoptosis of human polymorphonuclear neutrophils (PMN). Whereas TNF-alpha-mediated apoptosis was almost absent in the presence of the caspase-8 inhibitor Z-Ac-Ala-Glu-Val-Asp-7-fluoromethyl ketone (Z-AEVD-FMK), the inhibitor had no effect on spontaneous apoptosis, suggesting that spontaneous apoptosis was independent of caspase-8. Subsequently, we identified different isoforms of caspase-10 in human PMN and found high expression of caspase-10/b and/or -10/d and low expression of caspase-10/a and -10/c at the mRNA level. At the protein level, freshly isolated PMN showed high expression of caspase-10/b and -10/d as well as moderate expression of caspase-10/a and -10/c. Upon spontaneous apoptosis, caspase-10/b was down-regulated, which was accompanied by the appearance of a specific 47-kDa caspase-10/b cleavage product and an increased caspase-10 activity. In contrast, no down-regulation of caspase-10/a, -10/c, or -10/d was observed, suggesting that spontaneous apoptosis was associated with a differential activation of caspase-10/b. This was confirmed by the finding that spontaneous apoptosis was inhibited in the presence of Z-Ile-Glu-Thr-Asp (Z-IETD)-FMK, which blocks caspase-10. However, no down-regulation of caspase-10 isoforms was observed in the presence of TNF-alpha, suggesting that caspase-10 was not involved in TNF-alpha-induced apoptosis. Taken together, our study demonstrates that spontaneous and TNF-alpha-mediated apoptosis of PMN have different molecular requirements. Whereas TNF-alpha-mediated apoptosis depends on the activation of caspase-8, spontaneous apoptosis requires the activation of caspase-10/b. This finding may reveal that PMN apoptosis in different (patho-) physiological settings results from distinct molecular mechanisms.

Delay of neutrophil apoptosis in acute coronary syndromes.

Apoptosis of polymorphonuclear neutrophils (PMN) is currently discussed as a key event in the control of inflammation. This study determined PMN apoptosis and its underlying mechanisms in controls (C), patients with stable (SAP) or unstable angina (UAP), and with acute myocardial infarction (AMI). Blood was drawn from 15 subjects of each C, SAP, UAP, and AMI. Apoptosis was measured by flow cytometry in isolated PMN (propidium iodide staining) and PMN from whole blood (CD16, FcgammaRIII). Serum cytokines were determined by enzyme-linked immunosorbent assay. Apoptosis of isolated PMN was delayed significantly in acute coronary syndromes (ACS) as compared with SAP or C (C, 51.2+/-12.6%; SAP, 44.9+/-13.6%; UAP, 28.4+/-10.1%; AMI, 20.3+/-8.5%; AMI or UAP vs. SAP or C, P<0.001). These results were confirmed by measurement of PMN apoptosis in cultured whole blood from patients and controls. Moreover, serum of patients with ACS markedly reduced apoptosis of PMN from healthy donors. Analysis of patients' sera revealed significantly elevated concentrations of tumor necrosis factor alpha, interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin (IL)-1beta in ACS (vs. C and SAP). IFN-gamma, GM-CSF, and IL-1beta significantly delayed PMN apoptosis in vitro. Furthermore, coincubation of PMN with adenosine 5'-diphosphate-activated platelets significantly inhibited PMN apoptosis as compared with coculture with unstimulated platelets. This study demonstrates a pronounced delay of PMN apoptosis in UAP and AMI, which may result from increased serum levels of IFN-gamma, GM-CSF, and IL-1beta and from enhanced platelet activation. Therapeutical modulation of these determinants of PMN lifespan may provide a new concept for the control of inflammation in ACS.

Neutrophil Apoptosis is Inhibited in the Acute Phase of Kawasaki Disease

[Objectives] Circulating polymorphonuclear neutrophils(PMNs) are known to increase in number and are functionally activated in the acute phase of Kawasaki disease(KD). The aim of the present study is to investigate whether the apoptosis of PMNs is deregulated in acute KD. [Patients and methods] We studied the apoptosis of PMNs (AnnexinV-positive cells and cells with fragmented DNA) and the expression of Fas(CD95) and Bcl-2 protein(A1, Bax) using flow cytometer in KD patients(n=25), patients with a bacterial infection(BI, n=20), viral infection(VI, n=20) and healthy children(HC, n=20). [Results] When the isolated PMNs were cultured in vitro, the proportions of spontaneous apoptotic PMNs were found to be significantly lower (P<0.01) in the acute KD patients than in BI, VI or HC. The proportion of circulating Fas(CD95)-positive PMNs was also significantly lower (P<0.01) in the acute KD patients than in the other groups. In the acute phase of KD, the proportion of spontaneous apoptotic PMNs showed both a significant negative-correlation (P<0.01) with the peripheral PMN counts and a significant positive-correlation (P<0.01) with the proportions of circulating Fas(CD95)-positive PMNs. Furthermore, the agonistic anti-Fas mAb(CH-11) induced a significant increase in the proportion of apoptotic PMNs in the patients with a viral infection and healthy children, but not in either the patients with acute KD or the patients with a bacterial infection. In the intracellular expression of anti-(A1) and pro-apoptotic protein(Bax), the A1/Bax ratio was significantly higher in acute KD than in the other groups. [Conclusions] These findings indicate that PMN apoptosis is inhibited during the acute phase of KD and also suggest that both the resistance against the Fas-mediated death signal and the down-regulation of the mitochondrial apoptotic signaling pathway due to an altered balance of Bcl-2 protein expression are responsible for the delayed PMN apoptosis.

Mycobacterium tuberculosis-induced activation accelerates apoptosis in peripheral blood neutrophils from patients with active tuberculosis.

The activation of circulating polymorphonuclear neutrophils (PMN) from patients with active tuberculosis (TB-PMN) may be associated with induction of apoptosis. Spontaneous or Mycobacterium tuberculosis (MTB)-induced apoptosis of PMN were evaluated by microscopy, DNA content, and their binding to Annexin V at 0, 3, and 18 h. In addition, the expression of CD11b and of CD16 were evaluated as parameters of activation and apoptosis, respectively. Recently isolated TB-PMN showed a higher CD11b expression than normal PMN (N-PMN), but there were no features of apoptosis, even though an enhancement of Fas expression was observed. Spontaneous apoptosis was accelerated in TB-PMN at 3 h, but no differences were observed in TB- and N-PMN at 18 h of culture. When stimulated with MTB, both TB- and N-PMN steadily increased CD11b expression along the culture period. MTB induced apoptosis of N-PMN at 3 h with loss of CD16 expression. By contrast, MTB delayed the apoptotic rate of TB-PMN, preserving the CD16 receptor at 3 h, whereas it accelerated apoptosis at 18 h, increasing at the same time the expression of CD11b. Taken together, these data suggest that the acceleration of apoptosis observed in TB-PMN could be associated with the MTB-induced activation.

Extracellular matrix regulates apoptosis in human neutrophils

During inflammation, polymorphonuclear neutrophils (PMNs) migrate into the affected tissue interacting with extracellular matrix (ECM) proteins. We tested the hypothesis that PMN-matrix interaction affects PMN apoptosis. Apoptosis of human PMNs was detected by DNA-fragmentation assay and was quantitated by flow cytometry, ultraviolet and light microscopy. Cell adhesion was assessed by a toluidine blue assay, and cell spreading was detected by phase contrast microscopy. Protein tyrosine phosphorylation was studied using Western blotting and confocal microscopy. PMN apoptosis was not different in unstimulated cultures on either surface-adherent fibronectin or on PolyHema, a surface that prevents cell adherence. However, tumor necrosis factor-alpha (TNF alpha) treatment significantly increased apoptosis on fibronectin (37 +/- 4%) compared with PolyHema (20 +/- 3%). Tests on other matrix substances revealed that the percentage of apoptotic PMNs in the presence of TNF alpha was 8 +/- 1% on PolyHema, 26 +/- 4% on fibronectin, 17 +/- 2% on collagen I, 16 +/- 2% on collagen IV, and 16 +/- 3% on laminin (P < 0.05 for all matrices compared with PolyHema). Preincubation with genistein (50 microM) significantly inhibited TNF alpha-mediated apoptosis on fibronectin (39 +/- 4% to 21 +/- 4%) but not on PolyHema (21 +/- 4% to 16 +/- 4%). Genistein also reduced PMN spreading on fibronectin. In contrast, inhibitors of mitogen-activated protein kinase and protein kinase C showed no effect on PMN apoptosis. Fibronectin strongly increased tyrosine phosphorylation of three 102, 63, and 54 kDa proteins. Five newly tyrosine-phosphorylated 185, 85, 66, 56, and 42 kDa bands were also visible. Using confocal microscopy, highest tyrosine phosphorylation was localized to sites of cell-matrix interaction. ECM influences apoptosis in TNF alpha-activated, adherent, spreading PMNs. The process is regulated by tyrosine phosphorylation. Acceleration of apoptosis may shorten the PMN lifespan and thereby locally regulate inflammation.