Activation of neutrophil respiratory burst by cytokines and chemoattractants. Regulatory role of extracellular matrix glycoproteins

OBJECTIVE AND DESIGN: We investigated the in vitro responsiveness of neutrophils adherent to fibronectin (FN) and laminin (LM), toward natural pro-inflammatory and/or phagocyte-activating agents. MATERIALS AND METHODS: Neutrophils from normal volunteers were layered on polystyrene wells precoated or not with FN and/or LM and tested for their ability of responding to eleven pro-inflammatory mediators by evaluation of superoxide anion (O2-) production and adherence. Results, expressed as mean +/-1SEM, were evaluated by non-parametric analyses (Mann-Whitney U-test or Kruskal-Wallis non-parametric ANOVA analysis) RESULTS: Precoating polystyrene wells with LM or FN prevented the plastic-induced neutrophil (O2-) production. Among eleven agents, tumor necrosis factor-alpha (TNF, 3.0+/-0.3 nmoles (O2-)/5 x 10(4) neutrophils/180 min, p < 0.001), granulocyte-macrophage colony stimulating factor (GM-CSF, 2.1+/-0.3 nmoles (O2-)/5 x 10(4) neutrophils/180 min, p < 0.05) and formyl-peptides (fMLP, 2.5+/-0.5 nmoles (O2-)/5 x 10(4) neutrophils/180min, p < 0.01) caused massive (O2-) production by neutrophils adherent to FN. None of the mediators was capable of triggering (O2-) production by neutrophils adherent to LM. LM, mixed with FN to coat wells, caused a dose-dependent inhibition of the oxidative burst triggered by TNF (IC50 LM: 0.84+/-0.03 microg, mean+/-1 SEM), GM-CSF (IC50 LM: 0.36+/-0.16micro/g, mean+/-1SEM) and fMLP (IC50 LM: 0.54+/-0.008 microg, mean+/-1 SEM). To the contrary, fMLP (85.5+/-27.7%), TNF (163.1+/-67.5%), and GM-CSF (121.8+/-66.4%) caused a significant augmentation of neutrophil adherence to LM, suggesting that LM-mediated inhibition of neutrophil oxidative metabolism does not depend on the concomitant LM-induced inhibition of neutrophil adherence. Finally, neither solid-phase FN nor LM affected (O2-) production by neutrophils in response to immune complexes. CONCLUSIONS: Extracellular matrix glycoproteins dictate the response of neutrophils to soluble mediators but not to immune complexes. This appears to be a biologically meaningful mechanism to localise the risk of cellular reactions to mediators that are able to diffuse easily from tissue sites of generation and become widely distributed in body fluids during inflammatory diseases.     

Investigation of the role of nitric oxide and cyclic GMP in both the activation and inhibition of human neutrophils

1. The aim of this study was to establish the role of nitric oxide (NO) and cyclic GMP in chemotaxis and superoxide anion generation (SAG) by human neutrophils, by use of selective inhibitors of NO and cyclic GMP pathways. In addition, inhibition of neutrophil chemotaxis by NO releasing compounds and increases in neutrophil nitrate/nitrite and cyclic GMP levels were examined. The ultimate aim of this work was to resolve the paradox that NO both activates and inhibits human neutrophils. 2. A role for NO as a mediator of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis was supported by the finding that the NO synthase (NOS) inhibitor L-NMMA (500 microM) inhibited chemotaxis; EC50 for fMLP 28.76 +/- 5.62 and 41.13 +/- 4.77 pmol/10(6) cells with and without L-NMMA, respectively. Similarly the NO scavenger carboxy-PTIO (100 microM) inhibited chemotaxis; EC50 for fMLP 19.71 +/- 4.23 and 31.68 +/- 8.50 pmol/10(6) cells with and without carboxy-PTIO, respectively. 3. A role for cyclic GMP as a mediator of chemotaxis was supported by the finding that the guanylyl cyclase inhibitor LY 83583 (100 microM) completely inhibited chemotaxis and suppressed the maximal response; EC50 for fMLP 32.53 +/- 11.18 and 85.21 +/- 15.14 pmol/10(6) cells with and without LY 83583, respectively. The same pattern of inhibition was observed with the G-kinase inhibitor KT 5823 (10 microM); EC50 for fMLP 32.16 +/- 11.35 and > 135 pmol/10(6) cells with and without KT 5823, respectively. 4. The phosphatase inhibitor, 2,3-diphosphoglyceric acid (DPG) (100 microM) which inhibits phospholipase D, attenuated fMLP-induced chemotaxis; EC50 for fMLP 19.15 +/- 4.36 and 61.52 +/- 16.2 pmol/10(6) cells with and without DPG, respectively. 5. Although the NOS inhibitors L-NMMA and L-canavanine (500 microM) failed to inhibit fMLP-induced SAG, carboxy-PTIO caused significant inhibition (EC50 for fMLP 36.15 +/- 7.43 and 86.31 +/- 14.06 nM and reduced the maximal response from 22.14 +/- 1.5 to 9.8 +/- 1.6 nmol O2-/10(6) cells/10 min with and without carboxy-PTIO, respectively). This suggests NO is a mediator of fMLP-induced SAG. 6. A role for cyclic GMP as a mediator of SAG was supported by the effects of G-kinase inhibitors KT 5823 (10 microM) and Rp-8-pCPT-cGMPS (100 microM) which inhibited SAG giving EC50 for fMLP of 36.26 +/- 8.77 and 200.01 +/- 43.26 nM with and without KT 5823, and 28.35 +/- 10.8 and 49.25 +/- 16.79 nM with and without Rp-8-pCTP-cGMPS. 7. The phosphatase inhibitor DPG (500 microM) inhibited SAG; EC50 for fMLP 33.93 +/- 4.23 and 61.12 +/- 14.43 nM with and without DPG, respectively. 8. The NO releasing compounds inhibited fMLP-induced chemotaxis with a rank order of potency of GEA 3162 (IC50 = 14.72 +/- 1.6 microM) > GEA 5024 (IC50 = 18.44 +/- 0.43 microM) > SIN-1 (IC50 > 1000 microM). This order of potency correlated with their ability to increase cyclic GMP levels rather than the release of NO, where SIN-1 was most effective (SIN-1 (EC50 = 37.62 +/- 0.9 microM) > GEA 3162 (EC50 = 39.7 +/- 0.53 microM) > GEA 5024 (EC50 = 89.86 +/- 1.62 microM)). 9. In conclusion, chemotaxis and SAG induced by fMLP can be attenuated by inhibitors of phospholipase D, NO and cyclic GMP, suggesting a role for these agents in neutrophil activation. However, the increases in cyclic GMP and NO induced by fMLP, which are associated with neutrophil activation, are very small. In contrast much larger increases in NO and cyclic GMP, as observed with NO releasing compounds, inhibit chemotaxis.     

Activation of caspase 3 (CPP32)-like proteases is essential for TNF-alpha-induced hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in a murine endotoxin shock model

Endotoxin (ET)-induced liver failure is characterized by parenchymal cell apoptosis and inflammation leading to liver cell necrosis. Members of the caspase family have been implicated in the signal transduction pathway of apoptosis. The aim of this study was to characterize ET-induced hepatic caspase activation and apoptosis and to investigate their effect on neutrophil-mediated liver injury. Treatment of C3Heb/FeJ mice with 700 mg/kg galactosamine (Gal) and 100 microg/kg Salmonella abortus equi ET increased caspase 3-like protease activity (Asp-Val-Glu-Asp-substrate) by 1730 +/- 140% at 6 h. There was a parallel enhancement of apoptosis (assessed by DNA fragmentation ELISA and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay). In contrast, activity of caspase 1 (IL-1beta-converting enzyme)-like proteases (Tyr-Val-Ala-Asp-substrate) did not change throughout the experiment. Caspase 3-like protease activity and apoptosis was not induced by Gal/ET in ET-resistant mice (C3H/HeJ). Furthermore, only murine TNF-alpha but not IL-1alphabeta increased caspase activity and apoptosis. Gal/ET caused neutrophil-dependent hepatocellular necrosis at 7 h (area of necrosis, 45 +/- 3%). Delayed treatment with the caspase 3-like protease inhibitor Z-Val-Ala-Asp-CH2F (Z-VAD) (10 mg/kg at 3 h) attenuated apoptosis by 81 to 88% and prevented liver cell necrosis (< or = 5%). Z-VAD had no effect on the initial inflammatory response, including the sequestration of neutrophils in sinusoids. However, Z-VAD prevented neutrophil transmigration and necrosis. Our data indicate that activation of the caspase 3 subfamily of cysteine proteases is critical for the development of parenchymal cell apoptosis. In addition, excessive hepatocellular apoptosis can be an important signal for transmigration of primed neutrophils sequestered in sinusoids.     

Peroxynitrite-induced apoptosis in epithelial (T84) and macrophage (RAW 264.7) cell lines: effect of legume-derived polyphenols (phytolens)

Peroxynitrite (ONOO-) has been proposed as a mediator of gut inflammation and as an inducer of cell death by apoptosis. Phytolens (PHY), a water-soluble extract of polyphenolic antioxidants from nonsoy legumes (Biotics Research Corp, patent pending), was evaluated as a cytoprotective agent in human colonic (T84) and murine macrophage (RAW 264.7) cell lines. In the antioxidant testing, PHY showed a significant free radical scavenging ability against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and superoxide (O2.) radicals with an IC50 of 4.44 and 5.87 microg/ml against DPPH and O2., respectively. Apoptosis (DNA fragmentation) was measured by an ELISA technique. Cells were exposed to oxidative stress by treating them with peroxynitrite (100-300 microM) for 4 h in the presence and absence of PHY. Peroxynitrite elicited a dose-dependent increase in DNA fragmentation in both cell lines compared to the control group receiving decomposed ONOO-. PHY (10, 30, or 50 microg/ml) significantly attenuated the degree of apoptosis in T84 cells induced by ONOO- (P < 0.05). PHY (10-100 microg/ml) did not directly affect T84 cell viability or induce apoptosis after 4 h or overnight exposure. RAW 264.7 cells exposed to PHY alone (>30 microg/ml) for 4 h displayed decreased cell viability (P < 0.05) and increased apoptosis (P < 0.05). Phytolens may have beneficial effects on inflammation by attenuating peroxynitrite-induced apoptosis. The sparing of epithelial cells while compromising the viability of macrophages suggests that PHY may be beneficial in autoimmune disorders.     

Frequency-shaped amplification changes the neural representation of speech with noise-induced hearing loss

Apoptosis is a mode of active cell death. We have examined whether 2-chloroethylethyl sulfide (CEES), a sulfur vesicating agent, triggers apoptosis as a cytotoxic mechanism. Incubation of thymocytes with CEES, resulted in an induction of apoptotic features of cell death. Treatment of cells with 100 microM CEES for 5 h increased DNA fragmentation to approximately 40% of control. The fragmentation of DNA was visualized by agarose gel electrophoresis. It showed ladder pattern of DNA fragmentation, which indicates internucleosomal cleavage of DNA. Further evidence of apoptosis was observed in morphological changes of nuclei by using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. The percentage of TUNEL positive cells was dependent upon CEES concentrations. CEES induced the classical morphological features of apoptosis in nucleus. These features were accompanied by condensation of chromatin, which arranged in sharply declined clumps and fragmentation of nucleus. To study requirement for synthesis of new protein in CEES-induced apoptosis, we studied the effect of cycloheximide for apoptotic activity. This protein synthesis inhibitor did not suppress the CEES-induced apoptotic activity. Taken together, these results suggest that CEES-induced apoptosis as a cytotoxicmechanism and this process occurs independent of synthesis of new protein.     

The status and distance of cone biopsy margins as a predictor of excision adequacy for endocervical adenocarcinoma in situ

The purpose of this study was to begin to examine the influence of inhaled NO on O2 toxicity. The survival of Sprague-Dawley rats exposed to >95% O2, >95% O2 + 10 ppm NO, >95% O2 + 100 ppm NO, and >95% O2 + 3 ppm NO2 was determined. Survival at 120 h was 2/24 in >95% O2, 2/12 in >95% O2 + 10 ppm NO, and 1/12 in >95% O2 + 3 ppm NO2. Survival at 120 h was 21/30 in >95% O2 + 100 ppm NO (p < 0.01 compared with >95% O2). Three additional groups of rats were exposed for 60 h to: 21% O2, >95% O2, or >95% O2 + 100 ppm NO. The lungs were then assayed for total protein, reduced (GSH) and oxidized glutathione (GSSG), and 4-hydroxy-2(E)-nonenal. Both of the high O2 groups had significantly (p < 0.05) lower GSH/mg protein and GSH/GSSG ratios compared with the 21% O2 group. The >95% O2 group had a higher 4-hydroxy-2(E)-nonenal/mg of protein than either the 21% O2 group (p < 0.05), or the >95% O2 + 100 ppm NO group (p < 0.05 compared with >95% O2, not different from the 21% O2 group). Additional groups of rats were exposed to either 21% O2, >95% O2, or >95% O2 + 100 ppm NO for 0, 24, 48, and 60 h. The lungs were examined for neutrophil accumulation, which was increased at 60 h in the two groups exposed to >95% O2, but adding NO had no effect. Thus, the overall result was that 100 ppm inhaled NO improved the survival of rats in high O2.     

Early detection and control of risk factors in diabetic nephropathy

An influx of neutrophils into the airways is a common feature observed during pulmonary inflammation induced by air pollutants, including sulfur dioxide and sulfates. In the present study focusing on the in vitro interactions of sodium sulfite (Na2SO3) with human neutrophils, we confirm results indicating that this sulfite induces superoxide production (O2-) by itself. We demonstrated that this response can occur more rapidly than previously reported (within 5 min), and that Na2SO3 can act as a priming agent, in a concentration-dependent fashion, to the bacterial tripeptide N-formyl-methionine-leucine-phenylalanine (fMLP) by increasing O2-production. In addition, our results show that Na2SO3 induces gene expression in human neutrophils in a concentration-dependent manner as assessed by incorporation of 5-[3H] uridine into total RNA. However, it does not induce cell shape changes. We also demonstrated that Na2SO3 does not modulate neutrophil apoptosis nor reverse the well-known delaying effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on apoptosis. We conclude that Na2SO3 acts rapidly on neutrophil physiology, within a few minutes with respect to superoxide production, and a few hours (4 h) with respect to gene expression without altering a biological process such as the rate of apoptosis evaluated after a long period of incubation (20 h). We further conclude that Na2SO3-induced production of O2does not drive neutrophils to undergo apoptosis, a mechanism known to occur in other conditions. Therefore, the potential toxicity of Na2SO3 during pulmonary inflammation or lung-associated diseases may be related to its ability to induce superoxide production without altering neutrophil apoptosis rate.     

Histopathological analysis of rat mesentery as a method for evaluating neutrophil migration: differential effects of dexamethasone and pertussis toxin

In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration. The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 +/- 0.19 and Dex = 0.35 +/- 0.13 vs saline (S) = 2.85 +/- 0.59; fMLP: Ptx = 0.43 +/- 0.09 and Dex 0.01 +/- 0.01 vs S = 1.08 +/- 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 +/- 1.4 and Dex = 3.06 +/- 0.76 vs S = 15.94 +/- 3.97; fMLP: Ptx = 3.85 +/- 0.56 and Dex = 0.40 +/- 0.16 vs S = 7.15 +/- 1.17 neutrophils/field) induced by the two agonists in the rat mesentery. The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 microns), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient. The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 +/- 0.38 vs S = 4.20 +/- 1.01; fMLP: Dex = 0.25 +/- 0.11 vs S = 2.20 +/- 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue. In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 +/- 2.25 vs S = 4.20 +/- 1.01; fMLP: Ptx = 4.66 +/- 1.24 vs S = 2.20 +/- 0.34 neutrophils in the lumen/field). This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial space. In conclusion, the method described here is useful for quantifying the inflammatory and anti-inflammatory effect of different substances. The advantage of this histopathological approach is that it provides additional information about the steps involved in leucocyte migration.     

Cellular localization of the inhibitory action of abruquinone A against respiratory burst in rat neutrophils

1. The possible mechanisms of action of the inhibitory effect of abruquinone A on the respiratory burst in rat neutrophils in vitro was investigated. 2. Abruquinone A caused an irreversible and a concentration-dependent inhibition of formylmethionylleucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2.-) generation with IC50 values of 0.33 +/- 0.05 microgram ml-1 and 0.49 +/- 0.04 microgram ml-1, respectively. 3. Abruquinone A also inhibited O2 consumption in neutrophils in response to fMLP/CB and PMA. However, abruquinone A did not scavenge the generated O2.- in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation. 4. Abruquinone A inhibited both the transient elevation of [Ca2+]i in the absence of [Ca2+]o (IC50 7.8 +/- 0.2 micrograms ml-1) and the generation of inositol trisphosphate (IP3) (IC50 10.6 +/- 2.0 micrograms ml-1) in response to fMLP. 5. Abruquinone A did not affect the enzyme activaties of neutrophil cytosolic protein kinase C (PKC) and porcine heart protein kinase A (PKA). 6. Abruquinone A had no effect on intracellular guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels but decreased the adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 7. The cellular formation of phosphatidic acid (PA) and phosphatidylethanol (PEt) induced by fMLP/ CB was inhibited by abruquinone A with IC50 values of 2.2 +/- 0.6 micrograms ml-1 and 2.5 +/- 0.3 micrograms ml-1, respectively. Abruquinone A did not inhibit the fMLP/CB-induced protein tyrosine phosphorylation but induced additional phosphotyrosine accumulation on proteins of 73-78 kDa in activated neutrophils. 8. Abruquinone A inhibited both the O2.- generation in PMA-activated neutrophil particulate NADPH oxidase (IC50 0.6 +/- 0.1 microgram ml-1) and the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free system (IC50 1.5 +/- 0.2 micrograms ml-1) 9. Collectively, these results indicate that the inhibition of respiratory burst in rat neutrophils by abruquinone A is mediated partly by the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways, and by suppressing the function of NADPH oxidase through the interruption of electron transport.     

The effect of nitrogen dioxide exposure on the release of surfactant isolated from neonatal rabbit type II pneumocytes in culture

Nitrogen dioxide (NO2) is a well-known environmental air toxin, produced from a variety of sources, including cigarette smoke. Because of the growing knowledge of the harmful effects of passive smoking on children, we decided to study the effect of NO2 exposure on the release of surfactant from isolated neonatal type II pulmonary epithelial cells. After isolation from 1 to 4 day old rabbits, type II epithelial cells were allowed to adhere for 18 hours, washed, media changed, and were exposed to either 5% CO2 in room air or NO2, 5 ppm, for 2 hours (all results mean +/- sd; comparisons, paired t-test). There was no difference in cell number or viability prior to exposure. Cells exposed to NO2 had an increase in LDH release [LDH activity in media/(LDH in media+cells) x 100], air 12.6 +/- 2.2%, NO2 21.7 +/- 3.7%, (p < 0.05). NO2-exposed cells also had an increase in total phospholipid (microgram/cell culture dish) in media compared to air exposed, air 170.13 +/- 7.54, NO2 195.15 +/- 11.2, (p < 0.05). 3H-choline incorporation as a precursor to disaturated phosphatidylcholine (DSPC) was also conducted during exposure to either air or NO2. Incorporation of 3H-choline into surfactant lipid was increased in media from cells after NO2 exposure compared to air, 58.23 +/- 15.16 air, 76.81 +/- 19.86 NO2 (cpm/microgram protein; p < 0.05). These results show that 2 hours of 5 ppm NO2 exposure is associated with an increase in release of surfactant from neonatal type II cells in culture.     

Micromolar L-glutamate induces extensive apoptosis in an apoptotic-necrotic continuum of insult-dependent, excitotoxic injury in cultured cortical neurones

Excitotoxicity induced by L-glutamate (Glu), when examined in a pure neuronal cortical culture, involved widespread apoptosis at concentrations of 1-10 microM as part of a continuum of injury, which at its most servere was purely necrotic. Cells, maintained in chemically defined neurobasal/B27 medium, were exposed at d7 for 2 h to Glu (1-500 microM), and cellular injury was analysed 2 and 24 h after insult using morphology (phase-contrast microscopy), a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay, nuclear staining with 4,6-diamidino-2-phenylindole (DAPI), terminal transferase-mediated dUTP nick end-labelling (TUNEL) and DNA fragmentation by gel electrophoresis. Glu-mediated neurotoxicity was prevented by MK-801 (5 microM), whilst CNQX (20 microM) attenuated injury by 20%. Exposure to intensive insults (100 and 500 microM Glu) induced necrosis characterized by rapid cell swelling (< 2 h) and lack of chromatin condensation, confirmed by DAPI nuclear staining. In contrast, mild insults (< 20 microM Glu) failed to produce acute neuronal swelling at < 2 h, but 24 h after injury resulted in a large number of apoptotic nuclei as confirmed by TUNEL and electrophoretic evidence of DNA fragmentation, which was attenuated by cycloheximide (0.1 microg/ml). Our findings indicate for the first time that physiological concentrations of Glu produce neuronal injury across a continuum involving apoptosis (< 20 microM) and increasingly necrosis(> 20 microM), dependent on the severity of the initial insult.     

Involvement of platelet-activating factor in cell death induced under ischemia/postischemia-like conditions in an immortalized hippocampal cell line

The involvement of platelet-activating factor (PAF) in cell damage induced by ischemia/postischemia-like conditions was studied in a hippocampus-derived cell line, HN33.11. Cells exposed to N2-saturated glucose-free HEPES-buffered saline (ischemia) for 5 h followed by 18 h of incubation in serum-free control medium (postischemia reincubation) remained 67.4 +/- 2.4% viable in comparison with sham-treated cells. Analysis of DNA fragmentation in combination with Hoechst 33258 staining indicates that apoptosis is the dominant mode of cell death in the present model. PAF level during 10 h of ischemia was unchanged. However, an increase in PAF accumulation was found early during the reincubation period that followed 5 h of ischemia. Peak PAF concentrations were noted at 2 h after initiation of reincubation and rapidly declined to control level after 7 h of reincubation. Consistent with a role of PAF in mediating cell death under ischemia/postischemia reincubation in this model, the PAF antagonist BN 50739 exerted a dose-dependent protective effect. Maximal protection (85.7 +/- 5.4%) of the cells from ischemia/reincubation-induced cell damage was achieved at 0.1 microM BN 50739. The PAF antagonist lacked any protective effect against ischemia-induced cell death. On the other hand, the addition of the stable PAF analogue 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (MC-PAF) at the onset of ischemia potentiated ischemia/reincubation-induced apoptosis--an effect that was blocked by BN 50739. Pretreatment of HN33.11 cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) also provided a protective effect against ischemia/reincubation-induced cell damage. BAPTA-AM increased cell viability by 50%. Pretreatment with BAPTA-AM also decreased ischemia/reincubation-induced PAF accumulation in HN33.11 cells. The results suggest that PAF, acting via a PAF receptor, is at least in part mediating apoptosis under ischemia/postischemia-like conditions in HN33.11 cells.     

Inflammatory response during cardiopulmonary bypass: neutrophil apoptosis

Cardiopulmonary bypass (CPB) is essential to open heart surgery. However, CPB induces many types of inflammatory response, and may contribute to the tissue injury and the development of postoperative complications. On the other hand, the neutrophil responses to injury and infection immediately and secretes elastases and cytokines followed by prolongation of inflammatory changes, and programmed cell death (apoptosis) of neutrophils is delayed by inflammatory response. In this study, we evaluated the alternation of the neutrophil life span during CPB. Peripheral blood was obtained from eight adult patients before CPB, 1 hr and 2 hr after CPB start. After separation of neutrophils, and incuvation in the presence of TNF-alpha for 3 hr, we measured fluorescence-microscopically apoptosis rate (%A). %A significantly decreased with time (before 9.7 +/- 2.3%, 1 hr 3.0 +/- 1.0%, 2 hr 1.5 +/- 0.6%, p < 0.05). We conclude that neutrophil apoptosis was suppressed significantly during CPB. Systemic inflammatory change induced by CPB may be prolonged with extended life span of neutrophil.     

Effects of neutral endopeptidase inhibition and combined angiotensin converting enzyme and neutral endopeptidase inhibition on angiotensin and bradykinin peptides in rats

The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72 degrees C for 1 second. After exposure to temperatures of 58 to 59 degrees C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66 degrees C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72 degrees C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59 degrees C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation.     

Circulating mediators in serum of injured patients with septic complications inhibit neutrophil apoptosis through up-regulation of protein-tyrosine phosphorylation

BACKGROUND: The accumulation of neutrophils at inflammatory sites results in excessive release of toxic metabolites causing tissue injury. Proinflammatory cytokines may cause the breakdown of homeostasis of neutrophil numbers through inhibition of apoptosis. METHODS: Neutrophils were isolated from healthy humans and from patients with multiple injuries on day of admission and during septic complications. Apoptosis was quantitated using propidium iodide fluorescence and the TUNEL method. Tyrosine phosphorylation was measured by flow cytometry. RESULTS: Neutrophil apoptosis was decreased (33.3 +/- 5.5%; p < 0.05) in injured patients with sepsis compared with healthy humans (87.2 +/- 3.0%) and injured patients without sepsis (76.0 +/- 2.0%). Serum from injured patients with sepsis inhibited (p < 0.05) apoptosis of neutrophils from healthy humans in a dose-dependent manner. Serum from healthy humans and from injured patients at admission was ineffective. Neutralization of granulocyte-colony stimulating factor, but not of granulocyte-macrophage-colony stimulating factor, in serum of injured patients with sepsis partially abrogated (+51.2%) serum induced prolongation of neutrophil life span. Reduction of neutrophil apoptosis was concomitant with increased tyrosine phosphorylation. CONCLUSIONS: Septic complications, but not the injury itself, result in inhibition of spontaneous neutrophil apoptosis. Circulating mediators seem to reduce neutrophil apoptosis through up-regulation of tyrosine phosphorylation.     

Chronic exposure to kappa-opioids enhances the susceptibility of immortalized neurons (F-11kappa 7) to apoptosis-inducing drugs by a mechanism that may involve ceramide

Chronic exposure of embryonic brain to opioids leads to microcephaly and developmental abnormalities. An immortalized mouse neuroblastoma x dorsal root ganglion hybrid cell line stably transfected to overexpress kappa-opioid receptors (F-11kappa7) showed complete loss of kappa-receptor binding to [3H]U69,593 after exposure to the kappa-agonist U69,593 for 24 h. U69,593 had no measurable effect on cell viability as determined by either cell viability or DNA fragmentation assays. However, when cell death (apoptosis) was induced by either staurosporine or the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002, cells pretreated with U69,593 for 24 h showed increased apoptosis compared with untreated cells. Thus, staurosporine (50 nM), wortmannin (4 microM), and LY294002 (30 microM) treatment for 24 h induced a 50% loss of cell viability and DNA fragmentation in 24 h. U69,593 pretreatment produced the same killing at lower concentrations, namely, 20 nM staurosporine, 2 microM wortmannin, and 14 microM LY294002, respectively. The effects of U69,593 were time-, dose-, and naloxone-reversible, suggesting that they are receptor-mediated. However, coaddition of U69,593 at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. All three drugs that induced apoptosis were found to increase the level of ceramide, and pretreatment with U69,593 further increased the rate of formation of ceramide, a lipid that induces apoptosis in cells. We propose that chronic exposure to kappa-receptor agonists promotes increased vulnerability of neurons to apoptosis.     

Apoptosis and delayed neuronal damage after carbon monoxide poisoning in the rat

Delayed neurological damage after CO hypoxia was studied in rats to determine whether programmed cell death (PCD), in addition to necrosis, is involved in neuronal death. In rats exposed to either air or CO (2500 ppm), microdialysis in brain cortex and hippocampus was performed to determine the extent of glutamate release and hydroxyl radical generation during the exposures. Groups of control and CO-exposed rats also were tested in a radial maze to assess the effects of the CO exposures on learning and memory. At 3, 7, and 21 days after CO exposure brains were perfusion-fixed and hematoxylin-eosin (H&E) was used to assess injury and to select regions for further examination. DNA fragmentation was sought by examining cryosections with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) reaction. We found significant increases in glutamate release and .OH generation during and immediately after CO hypoxia. CO-exposed rats showed learning and memory deficits after exposure associated with heterogeneous cell loss in cortex, globus pallidus, and cerebellum. The frontal cortex was affected most seriously; the damage was slight at Day 3, increased at Day 7, and persistent at Day 21 after CO exposure. TUNEL staining was positive at all three time points, and TUNEL-labeled cells were distributed similarly to eosinophilic cells. The number of cells stained by TUNEL was less than by H&E and amounted to 2 to 5% of all cell nuclei in regions of injury. Ultrastructural features of both neuronal necrosis and apoptosis also were observed readily by electron microscopy. These findings indicate that both necrosis and apoptosis (PCD) contribute to CO poisoning-induced brain cell death.     

Changes of neutrophil migration without modification of in vitro metabolism and adhesion in Beh?et's disease

OBJECTIVE: Increase of neutrophil chemotaxis in Beh?et's disease (BD) has been described, but it is not clear whether there is a correlation with other variables of neutrophil function and whether these modifications correlate with disease activity. METHODS: We studied neutrophil functions in patients with BD in the acute phase in comparison with healthy control subjects and with the same patients during disease remission, with or without therapy. We investigated in vivo neutrophil migration by Senn's skin window technique and measured adhesion assay and superoxide production in circulating and migrating neutrophils after different stimuli. RESULTS: Neutrophil migration in vivo was 101.3 +/- 17.9 x 10(6) polymorphonuclear lymphocytes (PMN)/cm2/24 h in patients with BD in the acute phase and 66.1 +/- 7.8 x 10(6) PMN/cm2/24 h in controls (p < 0.001). No correlation was found between leukocyte counts and neutrophil migration. Neutrophil migration evaluated in the same patients in a phase of disease remission was 58.3 +/- 10.3 x 10(6) PMN/cm2/24 h (p < 0.001 vs acute phase, not significant vs controls). The neutrophils of the exudate were normally primed to response to the chemotactic peptide fMLP. No differences between the 2 groups were found in superoxide production, adhesion under basal conditions, or in response to different stimuli by circulating and migrating neutrophils. CONCLUSION: Abnormally high migration of neutrophils in the active phase of BD is the only consistent neutrophil dysfunction. Since this modification is reversed by therapy, the evaluation of in vivo neutrophil migration may be useful in diagnosing and monitoring disease activity. Blood neutrophils have normal responses to different stimuli, indicating they are not primed by the disease state.     

Low levels of hydrogen peroxide and L-histidine induce DNA double-strand breakage and apoptosis

The results presented in this study demonstrate that L-histidine triggers a lethal response in U937 cells exposed to nontoxic, albeit growth-inhibitory, levels of H2O2. Treatment for 1 h with the cocktail H2O2/L-histidine promotes the formation of a low level of DNA double-strand breaks that are rapidly rejoined, and this process is followed by secondary DNA fragmentation at about 7 h of post-treatment incubation, at which time cells are still viable. The appearance of oligonucleosomal DNA fragments associated with the detection of morphological changes typical of apoptosis strongly suggests that a portion of the cells was undergoing an apoptotic process. The relative level of cells with fragmented chromatin never exceeded 15-20% throughout the 20 h post-treatment incubation. Treatment with high concentrations of H2O2 in the presence of L-histidine was found to trigger necrotic cell death. The results presented in this paper provide further experimental evidence in support of the notion that DNA double-strand breaks mediate the lethal effects of the cocktail H2O2/L-histidine and suggest that this type of DNA lesion can promote both apoptotic and necrotic cell death, depending on the concentration of the oxidant.     

In vitro evaluation of neutrophil viability after exposure to a hypotonic medium

Separation techniques for radiolabelled leukocytes have inherent problems with contaminants (e.g. platelets and erythrocytes). Hypotonic lysis methods can eliminate the erythrocytes, but the question of neutrophil viability after an exposure to a hypotonic solution (i.e. sterile water) remains. Ficoll/ hypaque two-density gradient separation was performed on donor whole blood to obtain a pure neutrophil suspension. A timed sequence of water exposure was done for 5-100 s on the neutrophil preparations. The viability of these preparations was evaluated using flow cytometry and chemotaxis. The trypan blue staining method was used to document cell death. With water exposures ranging up to 100 s, 2.04 +/- 1.80% neutrophils exhibited cellular degradation by flow cytometry, and all samples demonstrated viable neutrophils by chemotaxis and trypan blue staining. The hypotonic medium exposure times for leukocyte separations should be less than 30 s for neutrophils to retain their viability by these in vitro techniques.