Polymorphonuclear Cell Apoptosis Research Articles

Novel Insights into Sporotrichosis and Diabetes.

Sporotrichosis is a type of zoonotic subcutaneous mycosis caused by different species of dimorphic fungus of the genus Sporothrix, and it is the most common form of subcutaneous mycosis in Latin America. Sporotrichosis is generally restricted to cutaneous and lymphatic tissue (i.e., localized forms), and involvement in the viscera (i.e., disseminated or disseminated cutaneous form) is uncommon, especially in the central nervous system. However, immunosuppression in individuals with diabetes mellitus can lead to the disseminated form of the disease due to a failure to eliminate the pathogen and poor infection treatment outcomes. Possible correlations between patients with diabetes and their greater susceptibility to disseminated cases of sporotrichosis include a decreased cytokine response after stimulation, increased oxidative stress, decreased chemotaxis, phagocytic activity, adhesion and rolling of neutrophils and monocytes/macrophages, and increased macrophage/monocyte and polymorphonuclear cell apoptosis. Therefore, this review highlights novel insights into diabetes and sporotrichosis by investigating how chronic inflammation affects and aggravates the infection, the possible causes of the greater susceptibility of Sporothrix sp. to hematogenous dissemination in immunocompromised patients, and the main alterations that this dissemination can cause.

Impact of anemia treatment with methoxy polyethylene glycol-epoetin beta on polymorphonuclear cells apoptosis in predialysis patients with chronic kidney disease.

Some data in literature indicate increased apoptosis of polymorphonuclear cells (PMNs) in chronic kidney disease (CKD), what seems to be connected with anemia. Erythropoiesis-stimulating agents, used in anemia treatment in CKD may affect cells apoptosis. Aim of this study was to investigate impact of anemia treatment with methoxy polyethylene glycol-epoetin beta (CERA) on PMNs apoptosis in predialysis patients with CKD. Percentage of early and late apoptotic PMNs was measured by flow cytometry based on annexin V and propidium iodide binding. CD90 (Fas), CD95L (FasL), CD16 and CD11b expression on PMNs were evaluated by flow cytometry after incubation with respective monoclonal antibody. Percentage of PMNs in early and late apoptosis in CKD patients before CERA treatment was significantly higher to control group, which was accompanied by significantly higher Fas and Fas-L expression and significantly lower expression of CD16. CERA treatment downregulated significantly percentage of early, apoptotic PMNs but percentage of late apoptotic cells did not change and was still significantly higher to control group. In all investigated groups we observed a significant negative correlation between hemoglobin concentration and percentage of apoptotic PMNs, as well as Fas and FasL expression and significant positive correlation between Hb and CD16 expression. Our results indicate that PMNs apoptosis is increased in predialysis patients with CKD and anemia treatment with CERA may diminish readiness of PMNs to undergo apoptosis. This antiapoptotic impact of anemia treatment with CERA seems to concern early apoptotic PMNs before they undergo to late, irreversible stage of apoptosis.

Characterization of LIC11207, a novel leptospiral protein that is recognized by human convalescent sera and prevents apoptosis of polymorphonuclear leukocytes

We report the study of a predicted outer-membrane leptospiral protein encoded by the gene lic11207. This protein is conserved in several pathogenic leptospiral strains but is absent in the saprophyte Leptospira biflexa. This putative outer-membrane protein has a domain of unknown function (DUF) 1565 found in several phylogenetically diverse bacteria and in the archaeon Methanosarcina acetivorans. The gene was cloned and expressed in Escherichia coli BL21 (SI) strain using the expression vector pDEST17. The 34 kDa recombinant protein was tagged with N-terminal hexahistidine and purified by metal-charged chromatography. The purified protein was used to assess: reactivity with human convalescent sera; in vivo expression; ability to activate endothelial cells (EC); and ability to modulate the apoptosis of polymorphonuclear cells (PMNs). The LIC11207 coding sequence was identified in vivo in the hamster renal tubules during experimental infection with Leptospira interrogans. The rLIC11207 showed significant antigenicity against human convalescent sera when compared with sera from healthy donors. The recombinant protein did not alter the surface expression of E-selectin or intercellular adhesion molecule 1 (ICAM-1) in EC and failed to induce the release of von Willebrand factor (vWF). Interestingly, rLIC11207 delayed apoptosis of PMNs suggesting a possible role of this protein during the infection.

Effect of hypertonic saline on apoptosis of polymorphonuclear cells

BackgroundThe function of polymorphonuclear (PMN) cells can be influenced by the choice of resuscitation fluids in hemorrhagic shock. Widespread interest in the use of hypertonic solutions for resuscitation has led to extensive investigation of their immune-modulating properties. Hypertonic saline (HTS) is known to modulate immune reactions, preventing the multiorgan failure mediated by immune reactions in trauma and hemorrhagic shock. PMN cells play a key role in such immune-mediated inflammatory processes, and HTS is believed to affect these PMN cells. However, how these events influence the actual event of apoptosis has not yet been described. Thus, in the present study, we aimed to investigate the differences in the apoptosis of PMN cells when exposed to isotonic and hypertonic environments and the temporal relations between the interval of administration of HTS after the stimulation of PMN cells. MethodsWhole blood was sampled from healthy volunteers, and the PMN cells were isolated. The isolated layer of PMN cells was washed twice with phosphate-buffered saline to yield the PMN cells. The number of cells was kept uniform, and an overall survival rate greater than 95% was maintained. After stimulation of the isolated PMN cells with N-formyl-methionyl-leucyl-phenylalanine, the PMN cells were allocated into 3 study groups (i.e., 1 isotonic group and 2 hypertonic groups with an osmolarity of 160 mM and 180 mM each). The extent of apoptosis was investigated in each group after culturing the PMN cells for 0, 1, 3, 6, 12, 15, 18, and 24 h. Depending on whether the PMN cells were stimulated with N-formyl-methionyl-leucyl-phenylalanine, they were also divided into stimulated and nonstimulated groups. In the stimulated group, the hypertonic environment was fostered immediately (HTS 0 h) and 6 h (HTS 6 h) after stimulation, which was accomplished after allocating the cells into an isotonic group (140 mM) and a hypertonic group (180 mM), according to the concentration of the culture medium. The PMN cells were then cultured at 37°C for 15 h with 5% carbon dioxide incubation. Each PMN suspension was labeled with Annexin V-fluorescein isothiocyanate and propidium iodide. Each sample underwent immediate flow cytometric analysis. PMN cells with high propidium iodide uptake were considered nonviable (necrotic). Among the viable PMN cells, those with no Annexin V uptake were considered normal and those with Annexin V uptake were considered apoptotic. ResultsDecreased apoptosis was observed in the PMN cells stimulated with N-formyl-methionyl-leucyl-phenylalanine. Increased apoptosis was observed in the stimulated PMN cells incubated in hypertonic condition compared with the cells incubated in isotonic condition. Early HTS administration demonstrated increased apoptosis compared with late administration. ConclusionsHTS treatment resulted in increased PMN apoptosis and an anti-inflammatory effect. Decreased apoptosis (prolonged lifespan) has been implicated in neutrophil-mediated tissue damage. HTS, by increasing the apoptosis of PMN cells, attenuates the postinjury inflammatory response. Also, early treatment with HTS was more efficient than delayed treatment.

Effects of Spermidine and p‐Cresol on Polymorphonuclear Cell Apoptosis and Function

Polymorphonuclear leukocytes (PMNs) from chronic kidney disease (CKD) patients display accelerated apoptosis and dysfunction, which may predispose CKD patients to infections. In this study, we investigated the effect of spermidine and p-cresol on apoptosis and function on PMN from healthy subjects. We measured the effect of spermidine and p-cresol on apoptosis, ROS production unstimulated and stimulated (S. aureus and PMA) and expression of CD95, caspase 3, and CD11b on PMN. After incubation with p-cresol and spermidine, we did not observe any changes in apoptosis, viability or expression of caspase 3 and CD95 in PMN from healthy subjects. PMN incubated for 10 minutes with spermidine demonstrated a significant reduction in spontaneous, S. aureus and PMA-stimulated ROS production. p-cresol induced a decrease in PMA-stimulated ROS production. Spermidine and p-cresol also induced a decrease in the expression of CD11b on PMN. Spermidine and p-cresol decreased the expression of CD11b and oxidative burst of PMN from healthy subjects and had no effect on PMN apoptosis and viability.

Molecular Pathways of Spontaneous and TNF-α–Mediated Neutrophil Apoptosis under Intermittent Hypoxia

Apoptosis of polymorphonuclear cells (PMNs) is a fundamental mechanism to halt inflammation. It limits the lifespan of PMNs and thereby decreases tissue injury. In PMNs, unlike in other cells, hypoxia profoundly inhibits apoptosis. However, most studies investigating hypoxic effects on the functioning of PMN focus on acute or chronic sustained hypoxia. Thus, the mechanisms by which intermittent hypoxia (IH) affects PMN apoptosis are not known. Flow cytometry and Western blotting were used to evaluate mechanisms of constitutive and TNF-α-mediated PMN apoptosis in IH. The levels of NF-κB, p38 mitogen-activated protein kinase (MAPK), TNF receptor-2 (TNFR-2), intracellular IL-8 and its surface receptor CXCR2, were determined. Specific NF-κB (gliotoxin and parthenolide) and p38MAPK (SB202190) inhibitors were also used. TNF-α-mediated PMN apoptosis was concentration-dependent; low concentration increased PMN survival, whereas higher concentrations accelerated apoptosis. However, at all TNF-α concentrations, PMN survival was higher after four IH cycles than in normoxia. However, increasing the IH cycles to six abolished the pro-apoptotic/anti-apoptotic effects of TNF-α. Also, IH increased TNRF2 expression, nuclear NF-κB translocation, p38MAPK phosphorylation, and expression of IL-8 and CXCR2. The NF-κB inhibitors gliotoxin and parthenolide increased apoptosis and decreased IL-8 and CXCR2 expression. Also, the p38MAPK inhibitor SB202190 increased apoptosis and decreased IL-8 expression but had no effect on CXCR2 expression. Collectively, these findings provide insights into the mechanisms that prolong PMN survival after IH exposure and demonstrate the essential role played by NF-κB, the p38MAPK signaling pathway, and downstream genes in this process.

Aprotinin Reduces Cardiac Troponin I Release and Inhibits Apoptosis of Polymorphonuclear Cells During Off-Pump Coronary Artery Bypass Surgery

In addition to blood-sparing effects, aprotinin may have cardioprotective and anti-inflammatory effects during cardiopulmonary bypass-assisted cardiac surgery. In this study, the authors examined whether aprotinin had cardioprotective and/or anti-inflammatory effects in patients undergoing off-pump coronary artery bypass grafting. A prospective randomized clinical trial. University hospital. Fifty patients were randomized to control (n = 25) or aprotinin treatment (n = 25) groups. Aprotinin was given as a loading dose (2 x 10(6) KIU) followed by a continuous infusion at 5 x 10(5) KIU/h until skin closure. Blood samples for cardiac troponin I; interleukin-6, interleukin-8, and interleukin-10; tumor necrosis factor alpha; and elastase were taken after anesthesia induction, completion of revascularization, and 6 hours, 12 hours, and 24 hours after revascularization. Blood samples were taken to assess for apoptosis in polymorphonuclear cells. Baseline plasma levels for cardiac troponin I did not differ between groups but were significantly lower in aprotinin-treated patients at the time of revascularization (p = 0.03) and 6 hours (p = 0.004) and 24 hours (p = 0.03) later. Aprotinin significantly reduced apoptosis in polymorphonuclear cells compared with control-treated patients (p = 0.04). There were no differences in plasma cytokine or elastase levels between groups. The authors conclude that aprotinin reduces perioperative cardiac troponin I release and attenuates apoptosis in polymorphonuclear cells but has no significant effects on plasma cytokine levels in patients undergoing off-pump coronary artery bypass graft surgery.

Effects of uraemia and dialysis modality on polymorphonuclear cell apoptosis and function

Previous studies have reported that incubation of polymorphonuclear cells (PMN) in uraemic plasma or with different haemodialysis membranes and peritoneal dialysis solutions increases apoptosis in this cell type. In addition, PMN harvested from uraemic patients show a reduced ability to generate superoxide in response to stimuli as well as impaired phagocytosis, chemotaxis and degranulation. The aim of the current study was to investigate the effect of uraemia and dialysis modality on apoptosis and function in freshly harvested non-incubated PMN. Polymorphonuclear cells were harvested from 14 chronic haemodialysis (HD) patients, from 14 continuous peritoneal dialysis patients (CAPD), 28 chronic kidney disease (CKD), pre-dialysis patients and from 14 healthy subjects (Controls). In these in vivo experiments, PMN apoptosis was studied by means of flow cytometric analysis of annexin V binding to freshly isolated cells. Polymorphonuclear cell phagocytosis and production of reactive oxygen species by unstimulated or stimulated (S.aureus, fMLP, PMA) cells were also studied by flow cytometry using whole blood. We observed increased PMN apoptosis in CKD patients. CAPD and HD patients displayed PMN apoptosis rates similar to controls. In the HD group, PMN exhibited decreased phagocytosis rates. In contrast, phagocytosis rates in PMN from CAPD were not significantly different from controls. In the CKD and HD groups, apoptosis was inversely correlated with respiratory burst activity and phagocytosis. Our results suggest that both uraemia and treatment modality may interfere with PMN apoptosis and function. Dialysis appears to normalize the increased PMN apoptosis rates observed in CKD patients.

In Vitro Effects of Polyamines on Polymorphonuclear Cell Apoptosis and Implications in the Pathogenesis of Periodontal Disease

Apoptosis provides a mechanism for clearance of unwanted cells in a variety of situations in which programmed or physiological cell death occurs; but the premature death of defensive cells could promote infection, inflammation and concomitant diseases. Polymorphonuclear cells (PMN) of gingival sulcus play an important role in host defense against periodontal tissue‐invading bacteria, but their phagocytic activity is conditioned by several virulence factors released by oral pathogens. Polyamines derived from oral bacteria frequently occur at concentrations approaching 1 mM in gingival fluid at diseased periodontal sites. Brief exposure of PMN to polyamines shortened the lag culture time required to observe microscopical or DNA fragmentation traces. Increase of Fas/Apo‐1 expression and caspase‐8 and caspase‐3 activation focused two typical steps in the pathway of the pro‐apoptotic mechanism exhibited by polyamines, even if to a different extent: spermine > spermidine > putrescine. The possible role played by polyamines in the pathogenesis of periodontal disease by dysregulating apoptosis of gingival PMN is discussed.

In vitro cytotoxicity assay to screen compounds for apoptosis-inducing potential on lymphocytes and neutrophils.

In vitro cytotoxicity assay to screen compounds for apoptosis-inducing potential on lymphocytes and neutrophils was investigated. Mouse, rat, dog, and human whole blood were incubated for 4 and 6 hr with actinomycin D, camptothecin, cortisone acetate, cycloheximide, doxorubicin, etoposide, 5-FU, mitomycin C and puromycin. Apoptotic lymphocytes and neutrophils were counted. All test compounds induced in vitro apoptosis of lymphocytes and/or neutrophils, but there were different potencies among the test compounds and there were also species differences in susceptibility. To investigate the in vivo effects of etoposide and cycloheximide which induced apoptosis of rat lymphocytes and that of rat lymphocytes and neutrophils, respectively, in in vitro assay, rats were intravenously administered either etoposide at 12.5, 25 or 50 mg/kg or cycloheximide at 1.25, 2.5 or 5 mg/kg. Etoposide caused decreases of circulating lymphocytes at 3 hr after administration in a dose-dependent manner, -16, -25 and -51%. Although cycloheximide caused neither decreased lymphocyte nor neutrophil counts, apoptosis in 30% of neutrophils was observed in rats receiving 5 mg/kg at 3 hr after administration. Etoposide at 50 mg/kg and cycloheximide at 5 mg/kg caused lymphocyte apoptosis in the spleen, thymus, mesenteric lymph nodes, bone marrow, and Peyer's patch from 1 to 6 hr after administration, with the maximum changes at 3 hr. In addition to apoptosis of these organs, cycloheximide at 5 mg/kg caused apoptosis of polymorphonuclear cells in the lamina propria of the small intestine. Therefore, it was found that the changes seen in the in vivo experiments considerably reflected the changes seen in the in vitro experiments. From these results, apoptosis is probably one of the major mechanisms for leukocyte toxicity induced by cytotoxic compounds, and the in vitro assay to screen compounds for acute apoptosis-inducing potential on lymphocytes and neutrophils would be useful as a primary screening method for animal toxicity studies. It may also be useful for risk assessments in advance of clinical trials.

Nicotine effects on polymorphonuclear cell apoptosis and lipopolysaccharide-induced monocyte functions. A possible role in periodontal disease?

Apoptosis provides a mechanism for clearance of unwanted cells in a variety of situations in which programmed or physiological cell death occurs; but the premature death of defensive cells could promote infection, inflammation and concomitant disease. We detected high values of apoptosis in polymorphonuclear cells (PMN) elicited from crevicular sulci of smokers affected by adult periodontitis. To learn more about the effects of nicotine on the periodontal environment, we studied its ability to modulate the apoptosis of two phagocytic lines, PMN and mononuclear cells, which are continuously recruited from gingival vessels to prevent or control plaque extension. Brief exposure of PMN to nicotine concentrations ranging from 0.01 to 0.3% shortened, in a dose-dependent relationship, the lag culture time required to observe at fluorescent microscopy the morphological traces of apoptosis. These observations were confirmed by specific tools of apoptosis: DNA fragmentation on gel electrophoresis and expression of the apoptosis-signaling receptor Fas/Apo-1. The apoptotic effect excited by nicotine on these first line defensive cells may be an important feature of the pathogenesis of periodontal disease. As for mononuclear leukocytes, nicotine was unable to induce apoptotic modifications on cells observed up to 72 h culture time, but the drug inhibited IL-1beta release and procoagulant activity (PCA) expression. The conflicting role played by these lipopolysaccharide (LPS)-induced monocyte functions in the inflammatory process is a further intrigue in the mechanism by which nicotine compromises the oral health.

Anti-Fcgamma receptor (FcgammaR) autoantibodies (Ab) delay apoptosis of polymorphonuclear cells (PMN) in systemic autoimmune diseases by inducing the production of G-CSF and GM-CSF

FcγRIIIb is expressed by PMN, and cell-free receptor has also been found circulating in body fluids. We have examined 222 patients with rheumatoid arthritis (RA), systemic lupus erythematosus and primary Sjogren's syndrome and classified anti-FcγR auto-Ab into three groups, based on the results of an indirect immunofluorescence (IIF) test and an ELISA : IIF+/ELISA+ (group A), IIF+/ELISA-(group B) and IIF-/ELISA+ (group C). PMN-binding Ab, i.e. groups A and B, prolonged the survival of PMN by delaying spontaneous apoptosis, and reduced adhesiveness and respiratory burst of these cells. Interestingly, recombinant non-glycosylated and purified glycosylated FcγR produced similar effects as the related auto-Ab. To delineate the mechanism(s) by which the PMN life span is prolonged, we studied the response of PMN to F(ab')2 fragments of anti-FcγR (CD16) monoclonal Ab. CD16 engagement decreased apoptosis and prevented the up-regulation of β2 integrins, particularly CD11b which is the α chain of complement receptor 3, and CD18 which is its β chain. The expression of mRNA for G-CSF and GM-CSF was induced. Release of these chimiokines followed CD16 stimulation, suggesting an autocrine involvement of survival factors in the rescue of PMN. The levels of G-CSF and GM-CSF paralleled the amounts of CD16 monoclonal Ab used to stimulate the cells. This set off a partial reduction of caspase 3 activity and was associated with down-expression of Bax. It was shown that anti-G-CSF and anti-GM-CSF Ab inhibited these events, while anti-TNFα was ineffective. Overall, apoptosis of aged PMN can be modulated by signalling through FcγR, which may occur in patients with PMN-binding auto-Ab. This might be particularly relevant in the synovial fluid of patients with RA.

Age-related effects of oxidative metabolism and cyclic AMP signaling on neutrophil apoptosis

Spontaneous as well as Fas-induced polymorphonuclear cell apoptosis is unchanged in the elderly. However, a weak responsiveness to antiapoptotic signals elicited by proinflammatory molecules has been reported in neutrophils isolated from aged humans. To gain insight into this field, here we have evaluated the role of oxidative metabolism and cyclic AMP (cAMP) signaling on age-related neutrophil apoptotic cell death. Results show that although superoxide dismutase (SOD), added exogenously to cell cultures, is able to prolong neutrophil survival in both young and aged individuals, high amounts of the enzyme are further effective in cell cultures of young donors only. Notably, the addition of catalase gives rise to a more striking, yet comparable, inhibition of neutrophil-programmed cell death in both groups of subjects. Furthermore, even low amounts of catalase are enough to restore a normal apoptotic outcome in SOD-treated cell cultures of old donors. Unlike the oxidative metabolism, cAMP signaling activation does not reveal any difference in the apoptotic response of neutrophils isolated from young and aged donors. Thus, supplementation of cell cultures with prostaglandin E2, dibutyryl cAMP or, to a lesser degree, forskolin results in a dose-dependent inhibition of DNA cleavage product appearance in both groups of subjects. The data outline that an impairment of neutrophil antioxidant shield, leading to an augmented cell oxidative load, is likely to occur as a feature of age. This may increase the apoptotic rate of stimulated cells, which may in turn account for the increased susceptibility of elderly individuals to life-threatening infections.

Polymorphonuclear cell apoptosis in exudates generated by polymers

Polymorphonuclear cell apoptosis in exudates generated by polymers

Necrosis and apoptosis of polymorphonuclear cells exposed to peritoneal dialysis fluids in vitro

Conventional peritoneal dialysis (PD) fluids are known to inhibit polymorphonuclear cells (PMN) phagocytosis, oxidative burst and enzyme release. However, the relative contributions of apoptosis and/or necrosis to this dysfunction have not been examined. We investigated the effects of osmolality, glucose concentration and heat-sterilization of PD fluids on necrosis and apoptosis of PMN. Polymorphonuclear cells were isolated from 8 healthy volunteers and exposed to different PD fluids for four hours. PMN were then double-stained with Hoechst 33342 and propidium iodide to study the proportion of viable, apoptotic and necrotic cells. Transmission electron microscopy (TEM) was performed to confirm the results obtained with flow cytometry. The fluids studied were conventionally heat-sterilized 1.5% Dianeal (1.5% D), conventionally heat-sterilized 4.25% Dianeal (4.25% D), 1.5% D in which the osmolality was increased to that of 4.25% D by adding mannitol (1.5% D + M), a filter-sterilized version of 4.25% D (4.25% D-F) and a 1.1% amino acid PD fluid (AA) (Nutrineal PD4). All PD fluids had their pH equilibrated (pH = 7.4) by the addition of sodium bicarbonate. Compared to PMN exposed to culture medium, a significantly higher proportion of necrosis was observed in PMN exposed to 1.5% D (P = 0.04). The 4.25% D induced greater necrosis than 1.5% D (P = 0.001), and the 4.25% D also induced significantly more necrosis (P = 0.002) compared to 4.25% D-F. These data suggest that the consequences of heat-sterilization, rather than high glucose concentration are responsible for the necrosis observed. Indeed, the proportion of necrotic PMN with 4.25% D-F was not significantly different from 1.5% D. The 1.5% D + M and AA induced significantly more apoptosis compared to 1.5% D (P = 0.006 and P < 0.05, respectively), suggesting that apoptosis can be induced by the high osmolality of PD fluids. However, 1.5% D +/- M also induced significantly more apoptosis (P = 0.007) compared to 4.25% D-F. This suggests that the apoptosis effect is specific for the osmolyte present in PD fluids, and that mannitol and amino acids induce more apoptosis than glucose. In summary, the different non-physiological components of conventional PD fluids evaluated in this study had a differential effect on PMN survival. Heat sterilization of high glucose-containing PD fluids was associated predominantly with necrosis of PMN, and high osmolality with apoptosis.