Polymorphonuclear Neutrophils Apoptosis Research Articles

Human Periodontal Stem Cells Release Specialized Proresolving Mediators and Carry Immunomodulatory and Prohealing Properties Regulated by Lipoxins.

Unresolved inflammation and tissue destruction are underlying mechanisms of periodontitis, which is linked to dysregulated polymorphonuclear neutrophil (PMN) functions. Lipoxin A4 (LXA4) is a specialized proresolving lipid mediator (SPM) that dampens excessive inflammation, promotes resolution, and protects from leukocyte-mediated tissue damage. Human periodontal ligament stem cells (hPDLSCs) represent key players during tissue regeneration and may contribute to resolution of inflammation; thus, they may represent a promising tool in regenerative dentistry. In the present study, we investigated the actions of hPDLSCs on PMN apoptosis and antimicrobial functions, and determined the impact of LXA4 on hPDLSCs. hPDLSCs significantly reduced apoptosis and stimulated microbicidal activity of human PMNs, via both cell-cell interactions and paracrine mechanisms. Lipid mediator metabololipidomics analysis demonstrated that hPDLSCs biosynthesize SPMs, including resolvin D1, D2, D5, and D6; protectin D1; maresins; and LXB4; as well as prostaglandins D2, E2, and F2α. LXA4 significantly enhanced proliferation, migration, and wound healing capacity of hPDLSCs through the activation of its cognate receptor ALX/FPR2, expressed on hPDLSCs. Together, these results demonstrate that hPDLSCs modulate PMN functions, and provide the first evidence that stem cells generate SPM and that the LXA4-ALX/FPR2 axis regulates regenerative functions of hPDLSCs by a novel receptor-mediated mechanism. These findings uncovered unappreciated features of stem cells from the periodontal ligament, supporting the notion that these cells may act as master regulators of pathophysiological events through the release of mediators that promote the resolution of inflammation and bacterial killing. The study also demonstrated that it is possible to modulate important functions of periodontal stem cells using lipoxin A4, a potent endogenous stop signal of inflammation. Thus, this study revealed an unappreciated anti-inflammatory proregenerative circuit that may be exploited to combat periodontal pathologies using resident stem cells. Moreover, the data may represent a more general template to explain the immunomodulatory functions of stem cells.

Delayed neutrophil apoptosis mediates intermittent hypoxia-induced progressive heart failure in pressure-overloaded rats.

Obstructive sleep apnea (OSA) and heart failure (HF) are common coexisting diseases. Intermittent hypoxia (IH), caused by repeated apnea/hypopnea events, accompanied by increased systemic inflammation, might contribute to the promotion of HF. To assess the hypothesis, rats were exposed to IH or normal air condition 4 weeks on the basis of normal heart function or pre-existing HF, which was induced by pressure overload caused by abdominal aortic constriction surgery performed 12 weeks earlier. Echocardiography was performed before and after IH exposure to evaluate left ventricular (LV) function. Serum concentrations of pro-inflammatory cytokines TNF-α and IL-6 were detected by enzyme-linked immunosorbent assay. Flow cytometric analysis was used to determine the apoptotic rate of polymorphonuclear neutrophils (PMNs). The echocardiographic study showed a significant decrease in LV fractional shortening (FS) and ejection fraction (EF) as well as an increase in the LV relative wall thickness (RWT) index in HF rats, which was aggravated by further exposure to IH compared with single-handed HF-only and sham-IH and sham-control groups. A reduced PMN apoptotic rate was observed in HF-IH rats compared with HF-only, sham-IH, and sham-control rats. Serum concentrations of TNF-α and IL-6 were also increased in HF-IH rats, accompanied by delayed PMN apoptosis, indicating significant systemic inflammation induced by IH. The results of this study demonstrated that IH aggravates LV remodeling and heart dysfunction in rats with pre-existing HF. Delayed neutrophil apoptosis, which was revealed in HF rats following exposure to IH, contributed to the exacerbation of myocardial damage and progression of heart dysfunction.

31 Sphingosine 1-phosphate receptor 3 is highly upregulated on human neutrophils upon migration to the airways

Objective Migration of polymorphonuclear neutrophils (PMNs) from blood into the airway lumen is accompanied by changes in surface receptors and responsiveness to environmental cues, such as sphingolipids. In particular, signaling via extracellular ceramide/sphingosine 1 phosphate (S1P) was suggested to contribute to human airway disease, such as cystic fibrosis (CF). We hypothesized that airway PMNs would alter expression of the S1P receptor, S1PR3, which was recently implicated in lung inflammation and fibrosis. Methods We collected blood and sputum from healthy control (HC) and CF subjects and analyzed S1PR3 expression in live PMNs gated by flow cytometry. In addition, we used a novel in vitro model to study the effect of transepithelial migration of blood PMNs on S1PR3 expression. Results We observed a 10-fold upregulation of surface S1PR3 expression on HC and CF airway PMNs, in vivo, compared to their blood counterparts. This major increase was recapitulated in vitro, when naive blood PMNs were induced to transmigrate through an airway epithelial layer upon exposure to apical CF airway fluid. S1PR3 expression increased over time, by 10-fold after 10 hours, and up to 20-fold at 18 hours. Conclusion Modulation of S1PR3 expression follows PMN migration to both HC and CF airways. Further studies will determine the importance of this receptor in mediating responses to ceramide/S1P signaling in these cells, including PMN migration, apoptosis and activation.

Sphingosine 1-phosphate receptor 3 is highly upregulated on human neutrophils upon migration to the airways (INC6P.315)

Abstract Background and hypothesis: Migration of polymorphonuclear neutrophils (PMNs) from blood into the airway lumen is accompanied by changes in surface receptors and responsiveness to environmental cues, such as sphingolipids. In particular, signaling via extracellular ceramide / sphingosine 1-phosphate (S1P) was suggested to contribute to human airway disease, such as cystic fibrosis (CF). We hypothesized that airway PMNs would alter expression of the S1P receptor, S1PR3, which was recently implicated in lung inflammation and fibrosis. Methods: We collected blood and sputum from healthy control (HC) and CF subjects and analyzed S1PR3 expression in live PMNs gated by flow cytometry. In addition, we used a novel in vitro model to study the effect of transepithelial migration of blood PMNs on S1PR3 expression. Results: We observed a 10-fold upregulation of surface S1PR3 expression on HC and CF airway PMNs, in vivo, compared to their blood counterparts. This major increase was recapitulated in vitro, when naive blood PMNs were induced to transmigrate through an airway epithelial layer toward apical CF airway fluid. S1PR3 expression increased over time, by 10-fold after 10 hours, and up to 20-fold at 18 hours. Conclusions: Modulation of S1PR3 expression follows PMN migration to both HC and CF airways. Further studies will determine the importance of this receptor in mediating responses to ceramide/S1P signaling in these cells, including PMN migration, apoptosis and activation.

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.

Regulatory mechanism of pyrrolidine dithiocarbamate is mediated by nuclear factor-κB and inhibits neutrophil accumulation in ARDS mice.

The aim of the present study was to investigate the regulatory mechanism of nuclear factor (NF)-κB on polymorphonuclear neutrophil (PMN) accumulation and the inflammatory response in lung tissues with acute respiratory distress syndrome (ARDS), as well as the therapeutic effect of pyrrolidine dithiocarbamate (PDTC). Mouse models of ARDS were established by intraperitoneal injection of lipopolysaccharide (LPS). BALB/c mice were divided into control, LPS and PDTC + LPS groups. The expression of PMN adhesion molecules, CD11b/CD18 and intercellular adhesion molecule-1 (ICAM-1), were detected by immunohistochemistry, while the protein expression levels of NF-κB p65 in the lung tissue were analyzed by western blot analysis. In addition, flow cytometry was used to investigate the apoptosis rate of PMNs in the bronchoalveolar fluid, and the expression levels of interleukin (IL)-1β, IL-8 and tumor necrosis factor (TNF)-α and myeloperoxidase (MPO) activity were also determined. Following an intraperitoneal injection of LPS, alveolar septum rupture, pulmonary interstitial hyperemia and PMN infiltration in the alveolar was observed. The protein expression of p65 in the pulmonary cytoplasm decreased, while the expression of p65 in the nucleus increased. The levels of IL-8, IL-1β and TNF-α increased and the high expression status was maintained for 24 h. As the time increased, CD11b/CD18 and ICAM-1 expression increased, as well as MPO activity, while the apoptosis of PMNs was delayed. Compared with the LPS group, the expression of p65 in the pulmonary cytoplasm and the PMN apoptosis rate increased following PDTC intervention, while the expression of p65 in the nucleus decreased, as well as the expression levels of the cytokines and MPO activity. Therefore, PDTC reduced the production of inflammatory cytokines via the NF-κB pathway, which reduced the activation of PMNs in the lung tissue and promoted PMN apoptosis.

L-Carnosine Modulates Respiratory Burst and Reactive Oxygen Species Production in Neutrophil Biochemistry and Function: May Oral Dosage Form of Non-Hydrolized Dipeptide L-Carnosine Complement Anti-Infective Anti-Influenza Flu Treatment, Prevention and Self-Care as an Alternative to the Conventional Vaccination?

Influenza A is a viral disease of global dimension, presenting with high morbidity and mortality in annual epidemics, and in pandemics which are of infrequent occurrence but which have very high attack rates. Influenza vaccines of the future must be directed toward use of conserved group-specific viral antigens, such as are present in transitional proteins which are exposed during the fusion of virus to the host cell. Influenza probes revealed a continuing battle for survival between host and parasite in which the host population updates the specificity of its pool of humoral immunity by contact with and response to infection with the most recent viruses which possess altered antigenic specificity in their hemagglutinin (HA) ligand. It is well known that the HA protein is found on the surface of the influenza virus particle and is responsible for binding to receptors on host cells and initiating infection. Polymorphonuclear neutrophils (PMN) have been reported to be involved in the initial host response to influenza A virus (IAV). Early after IAV infection, neutrophils infiltrate the airway probably due to release of chemokines that attract PMN. Clearly, severe IAV infection is characterized by increased neutrophil influx into the lung or upper respiratory tract. Carnosine (β-alanyl-L-histidine) and anserine (N-β-alanyl-1-methyl-L-histidine) are found in skeletal muscle of most vertebrates, including those used for food; for example, 100 g of chicken breast contains 400 mg (17.6 mmol/L) of carnosine and 1020 mg (33.6 mmol/l) of anserine. Carnosine-stimulated respiratory burst in neutrophils is a universal biological mechanism of influenza virus destruction. Our own studies revealed previously unappreciated functional effects of carnosine and related histidine containing compounds as a natural biological prevention and barrier against Influenza virus infection, expand public understanding of the antiviral properties of imidazole-containing dipeptide based compounds, and suggest important interactions between neutrophills and carnosine related compounds in the host response to viruses and bacteria. Carnosine and anserine were also found to reduce apoptosis of human neutrophils. In this way these histidine-containing compounds can modulate the Influenza virus release from neutrophills and reduce virus dissemination through the body of the organism. This review points the ability of therapeutic control of Influenza viral infections associated with modulation by oral nonhydrolized forms of carnosine and related histidine-containg compounds of PMN apoptosis which may be involved at least in part in the pathophysiology of the disease in animals and humans. The data presented in this article, overall, may have implications for global influenza surveillance and planning for pandemic influenza therapeutic prevention with oral forms of non-hydrolized natural L-carnosine as a suitable alternative to the conventional vaccination for various flu ailments.

Nicotinamide Phosphoribosyltransferase Inhibitor Is a Novel Therapeutic Candidate in Murine Models of Inflammatory Lung Injury

We previously identified the intracellular nicotinamide phosphoribosyltransferase (iNAMPT, aka pre-B-cell colony enhancing factor) as a candidate gene promoting acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI) with circulating nicotinamide phosphoribosyltransferase potently inducing NF-κB signaling in lung endothelium. iNAMPT also synthesizes intracellular nicotinamide adenine dinucleotide (iNAD) in response to extracellular oxidative stress, contributing to the inhibition of apoptosis via ill-defined mechanisms. We now further define the role of iNAMPT activity in the pathogenesis of ARDS/VILI using the selective iNAMPT inhibitor FK-866. C57/B6 mice were exposed to VILI (40 ml/kg, 4 h) or LPS (1.5 mg/kg, 18 h) after osmotic pump delivery of FK-866 (100 mg/kg/d, intraperitoneally). Assessment of total bronchoalveolar lavage (BAL) protein, polymorphonuclear neutrophil (PMN) levels, cytokine levels (TNF-α, IL-6, IL-1α), lung iNAD levels, and injury scores revealed that FK-866-mediated iNAMPT inhibition successfully reduced lung tissue iNAD levels, BAL injury indices, inflammatory cell infiltration, and lung injury scores in LPS- and VILI-exposed mice. FK-866 further increased lung PMN apoptosis, as reflected by caspase-3 activation in BAL PMNs. These findings support iNAMPT inhibition via FK-866 as a novel therapeutic agent for ARDS via enhanced apoptosis in inflammatory PMNs.

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.

Increased apoptosis of peripheral blood neutrophils is associated with reduced incidence of infection in trauma patients with hemorrhagic shock

We aimed to describe the relationship between early peripheral leukocyte apoptosis and incidence of subsequent infection in trauma patients with hemorrhagic shock (T/HS). T/HS patients requiring emergency surgery were prospectively enrolled. Nucleosome ELISA and TUNEL staining were performed on peripheral blood drawn pre-operatively, post-operatively and at 24 h. Subjects were followed for 30 days or until death or hospital discharge to record all episodes of infection. Forty-one subjects were enrolled. Six died within 24 h of surgery and were not included in the analysis. Nucleosome levels peaked post-operatively and dropped to baseline levels at 24 h (p = 0.03). TUNEL analysis revealed that polymorphonuclear neutrophils (PMNs) accounted for 72% of apoptotic leukocytes; the remaining apoptotic cells were mainly lymphocytes. Increased post-operative leukocyte apoptosis was associated with decreased systemic inflammatory response syndrome (SIRS) severity. Seventeen of the 35 survivors (48.6%) developed infections, while 18 (51.4%) did not. Pre-operative and post-operative nucleosome levels were 2.5 and 3 times higher, respectively, in T/HS patients who did not develop infection compared to those who did. Increased nucleosome levels were associated in particular with protection against sepsis (p=0.03) and multiple infections (p = 0.01). Peripheral blood PMN apoptosis in the early resuscitative period is associated with decreased incidence of subsequent infection in T/HS patients.

Cyclin-Dependent Kinase Inhibition Reduces Lung Damage in a Mouse Model of Ventilator-Induced Lung Injury

Mechanical ventilation (MV) has the potential to induce lung damage in healthy lungs or aggravate existing lung injury. Polymorphonuclear neutrophil (PMN) recruitment plays an important role in driving the inflammatory response in ventilator-induced lung injury (VILI). The cyclin-dependent kinase inhibitor r-roscovitine has been shown to induce apoptosis in PMNs. In this study, we investigated the potential of r-roscovitine treatment in reducing lung damage in a mouse model of VILI. Mice were tracheotomized and subjected to lung-protective MV with lower (∼7.5 mL/kg) or lung-injurious MV with higher (∼15 mL/kg) tidal volume (VT). R-roscovitine treatment enhanced apoptosis in PMNs in vitro. Ventilator-induced lung injury was associated with pulmonary PMN influx in low and high VT MV. During lung-injurious MV, r-roscovitine treatment reduced the number of PMNs and lowered levels of the lung damage markers RAGE (receptor for advanced glycation end products) and total immunoglobulin M in bronchoalveolar lavage fluid. R-roscovitine did not affect cytokine or chemokine levels in the bronchoalveolar space, neither during lung-protective nor lung-injurious MV. Thus, r-roscovitine treatment reduces lung damage in VILI, possibly dependent on increased apoptosis of PMNs.

Sulfur dioxide attenuates LPS-induced acute lung injury via enhancing polymorphonuclear neutrophil apoptosis

We speculated that the enhanced apoptosis of polymorphonuclear neutrophil (PMN) might be responsible for the inhibition of PMN infiltration in the lung. This study was designed to investigate the effects of sulfur dioxide (SO(2)) on PMN apoptosis in vivo and in vitro, which may mediate the protective action of SO(2) on pulmonary diseases. Acute lung injury (ALI) was induced by intratracheally instillation of lipopolysaccharide (LPS, 100 μg/100 g, in 200 μL saline) in adult male SD rats. SO(2) solution (25 μmol/kg) was administered intraperitoneally 30 min before LPS treatment. The rats were killed 6 h after LPS treatment. Lung tissues were collected for histopathologic study and SO(2) concentration assay. Bronchoalveolar lavage fluid (BALF) was collected for the measurement of PMN apoptosis. For in vitro experiments, rat peripheral blood PMNs were cultured and treated with LPS (30 mg/L) and SO(2) (10, 20 and 30 μmol/L) for 6 h, and apoptosis-related protein expression was detected by Western blotting, and apoptosis rate was measured with flow cytometry. LPS treatment significantly reduced the SO(2) concentrations in the lung tissue and peripheral blood, as compared with the control group. Pretreatment with SO(2) prevented LPS-induced reduction of the SO(2) concentration in the lung tissue and peripheral blood. LPS treatment significantly reduced PMN apoptosis both in vivo and in vitro, which could be prevented by the pretreatment with SO(2). The protein levels of Caspase-3 and Bax was significantly increased, but Bcl-2 was decreased by the pretreatment with SO(2), as compared with LPS administration alone. SO(2) plays an important role as the modulator of PMN apoptosis during LPS-induced ALI, which might be one of the mechanisms underlying the protective action of SO(2) on pulmonary diseases.

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.

Peripheral Blood Polymorphonuclear Neutrophils Apoptosis in Active and Latent Tuberculosis

Peripheral Blood Polymorphonuclear Neutrophils Apoptosis in Active and Latent Tuberculosis

Effects of 4-aminosalicylic acid on migration, activation and apoptosis of polymorphonuclear neutrophil in rats with experimental colitis

Objective To investigate the effects of 4-aminosalicylic acid (4-ASA) on rats with 2,4,6-trinitrobenzenesulfonic acid-induced colitis in order to understand its mechanisms in treatment of inflammatory bowel disease (IBD). Methods Thirty SD rats were given 2,4,6-trinitrobenzenesulfonic acid to induce colitis and were divided into model group, 5-ASA (200 mg/kg) treated group and 4-ASA (200 mg/kg) treated group with 10 each. Another 10 rats were severed as normal control. Seven days later,all animals were sacraficed for estimation of colonic tissues. The iNOS and serum level of interleukin (IL)-8 were detected by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) ,respectively. And the apoptosis of polymorphonuclear neutrophil (PMN) was examined by flow cytometry. Results In comparison with model group, the body weight was increased in rats treated with 4-ASA (t= 14.09,P 0. 05). The expression of tissue iNOS was 73.55%±5.15% in model group, which was higher than that in control group [(5.95±1.45)% ,t=39.93,P<0.01)],but was lower than that in 5-ASA treated group [(37.80±3.82)%,t = 17.62,P<0.01] and 4-ASA treated group [(42.27±3.52) %, t= 15.76 ,P<0.01]. The serum level of IL-8 in model group was significantly higher than that in 5-ASA treated group and 4-ASA treated group (P<0. 01). The apoptosis of PMN in model group was lower than that in control group (t= 11.48,P<0.01), but higher than that in 5-ASA treated group (t= 7.51, P<0.01) and 4-ASA treated group (t= 10.47,P<0.01). Conclusions The efficacy of 4-ASA in treatment of IBD may be related to the mechanisms of reducing the migration and the activities of PMN, up-regulating PMN apoptosis and scavenging reactive oxygen radicals produced by PMN. Key words: Inflammatory bowel disease; p-aminosalicylic acid; Interleukin-8; Apoptosis

Cytokines modulate neutrophil death

Polymorphonuclear neutrophils (PMN) are terminally differentiated cells with a short life span, in the blood circulation. The prolongation of the life span of PMN is critical in their effectiveness against pathogens, in particular in the tissues. This review summarizes the effect of cytokines on PMN apoptosis and points to some examples of pathological situations characterized by inappropriate cytokine production associated with dysregulation of PMN apoptosis.

In Cystic Fibrosis Homozygotes and Heterozygotes, Neutrophil Apoptosis Is Delayed and Modulated by Diamide or Roscovitine: Evidence for an Innate Neutrophil Disturbance

Cystic fibrosis (CF) is a chronic inflammatory lung disease characterized by polymorphonuclear neutrophil (PMN)-dominated airway inflammation. Defective apoptosis might explain PMN persistence at these inflammation sites. We previously reported that in CF patients PMN underwent delayed apoptosis, which was not always related to their infectious state and independent of the type of CF transmembrane regulator (CFTR) mutation. To understand the role of infection and PMN apoptosis in CF, PMN apoptosis was investigated in CF parents who are obligate heterozygotes for the CFTR mutation but without chronic bacterial infection. They also demonstrated delayed PMN apoptosis compared with healthy controls, as assessed by annexin-V labeling and caspase-3 cleavage. Diamide, a direct thiol-oxidizing agent, potentiated PMN apoptosis in controls and CF patients, resulting in similar levels of constitutive and Fas-potentiated apoptosis. The cyclin-dependent kinase inhibitor roscovitine provided another approach to restore normal PMN apoptosis. However, the selective CFTR inhibitor CFTR_Inh172 did not affect PMN apoptosis in control subjects. Apparently, the dysregulation of CF PMN is not only a consequence of the chronic infectious state in CF children but might also be related to CF ‘intrinsic’ factors. Restoration of normal PMN apoptosis by cellular redox modulation or roscovitine opens new research avenues to decrease PMN-mediated inflammation in CF.

Evidence that endoplasmic reticulum (ER) stress and caspase-4 activation occur in human neutrophils

Apoptosis can result from activation of three major pathways: the extrinsic, the intrinsic, and the most recently identified endoplasmic reticulum (ER) stress-mediated pathway. While the two former pathways are known to be operational in human polymorphonuclear neutrophils (PMNs), the existence of the ER stress-mediated pathway, generally involving caspase-4, has never been reported in these cells. Recently, we have documented that arsenic trioxide (ATO) induced apoptosis in human PMNs by a mechanism that needs to be further investigated. In this study, using immunofluorescence and electron microscopy, we present evidence of ER alterations in PMNs activated by the ER stress inducer arsenic trioxide (ATO). Several key players of the unfolded protein response, including GRP78, GADD153, ATF6, XBP1 and eIF2α are expressed and activated in PMNs treated with ATO or other ER stress inducers. Although caspase-4 is expressed and activated in neutrophils, treatment with a caspase-4 inhibitor did not attenuate the pro-apoptotic effect of ATO at a concentration that reverses caspase-4 processing and activation. Our results demonstrate for the first time that the ER stress-mediated apoptotic pathway operates in human neutrophils.

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 effect of different oxygenators on polymorphonuclear neutrophil apoptosis of heart surgery undergoing cardiopul-monary bypass

Objective To evaluate the effect of two kind oxygenators on polymorphonuclear neutrophil (PMN) apoptosis, and to give a method of inhibting the systemic inflammatory response syndrome(SIRS) to cardiopulmonary bypass(CPB). Methods Sixty VSD patients undergoing open heart surgery with CPB were randomly divided into two groups(n = 30): group B(bubble oxygenator), group M(membrane oxygenator). Blood samples were drawn from the central venous line before starting CPB, at the end of CPB, 4, 8 and 24 h after CPB. The PMN counts were performed by blood cell counter. PMN apoptosis and the expression of CD11b were evaluated by flow eytometry. The plasma concentration of elastase was determined by enzyme linked immunosorbent assays. Results The rates of PMN apoptosis were significantly reduced (P<0.05) and the PMN counts, the expression of CD11b and the plasma con-centration of elastase were remarkably increased after CPB(P<0.05), but at the end of CPB, 4 h and 8 h after CPB the rate of PMN apoptosis was higher in group M than that in group B. PMN counts, the expression of CDI lb and the plasma concentration of elastase were higher in group B than in group M (P<0.05). The PMN counts were higher in group B than in group M 24 h after CPB (P< 0.05). Conclusion XiJian-Ⅱ membrane oxygenator can increase PMN apoptosis and depress systemic inflammatory response better than XIJing-90 bubble oxygenator. Key words: Cardiopulmonary bypass; Inflammatory response; Membrane oxygenators; Bubble oxygenators