Showing papers on "Neutrophil extracellular traps published in 2004"

Neutrophil extracellular traps kill bacteria

Abstract: Neutrophils engulf and kill bacteria when their antimicrobial granules fuse with the phagosome. Here, we describe that, upon activation, neutrophils release granule proteins and chromatin that together form extracellular fibers that bind Gram-positive and -negative bacteria. These neutrophil extracellular traps (NETs) degrade virulence factors and kill bacteria. NETs are abundant in vivo in experimental dysentery and spontaneous human appendicitis, two examples of acute inflammation. NETs appear to be a form of innate response that binds microorganisms, prevents them from spreading, and ensures a high local concentration of antimicrobial agents to degrade virulence factors and kill bacteria.

The periplasmic serine protease inhibitor ecotin protects bacteria against neutrophil elastase.

Abstract: proteases Factor Xa, thrombin and urokinase-type plasminogen activator. To test whether ecotin does, in fact, protect bacteria from neutrophil elastase, an ecotin-deficient strain was generated in E. coli. This strain is significantly more sensitive in cell-killing assays to human neutrophil elastase, which causes increased permeability of the outer membrane that persists even during renewed bacterial growth. Ecotin affects primarily the ability of E. coli to recover and grow following treatment with neutrophil elastase, rather than the actual rate of killing. This suggests that an important part of the antimicrobial mechanism of neutrophil elastase may be a periplasmic bacteriostatic effect of protease that has translocated across the damaged outer membrane.

The Role of Interleukin-1β in Direct and Toll-Like Receptor 4-Mediated Neutrophil Activation and Survival

Abstract: The regulation of systemic and local neutrophil activation is crucial to the clearance of infections and the successful resolution of inflammation without progress to tissue damage or disseminated inflammatory reactions. Using purified lipopolysaccharide (pLPS) and highly purified neutrophils, we have previously shown that Toll-like receptor 4 signaling is a potent neutrophil activator, but a poor stimulator of survival. In the presence of peripheral blood mononuclear cells (PBMCs), however, pLPS becomes a potent neutrophil survival factor. Interleukin (IL)-1β has been identified as an important neutrophil activator and prosurvival cytokine, and is produced in abundance by LPS-stimulated PBMCs. We now show that IL-1β fails to activate highly purified neutrophils or enhance their survival, but in the presence of PBMCs, IL-1β induces neutrophil survival. We hypothesized that LPS-primed neutrophils might become responsive to IL-1β, but were unable to demonstrate this. Moreover, IL-1ra failed to prevent pLPS + PBMC-dependent neutrophil survival. In studies of IL-1R1−/− mice, we found that LPS was still able to mediate neutrophil survival, and neutrophil survival was enhanced by the addition of monocytic cells. Thus an important paradigm of neutrophil regulation needs to be viewed in the context of a cellular network in which actions of IL-1β on neutrophils are indirect and mediated by other cells.

Linkage of neutrophil serine proteases and decreased surfactant protein-A (SP-A) levels in inflammatory lung disease

Abstract: Background: In patients with cystic fibrosis (CF) neutrophils are recruited in excess to the airways yet pathogens are not cleared and the patients suffer from chronic infections. Recent studies have shown a deficiency in airway fluids from patients with CF and other inflammatory pulmonary conditions of surfactant protein A (SP-A), a pattern recognition molecule that facilitates uptake of microbes by macrophages and neutrophils. Methods: In vitro simulations were used to test the hypothesis that decreased SP-A levels in CF might be the result of degradation by neutrophil serine proteases. Results: Very low levels of the neutrophil granule serine proteases cathepsin G, elastase, and proteinase-3 rapidly degraded pure SP-A when tested in buffered saline. The order of potency was cathepsin G>elastase>proteinase-3. The addition of cathepsin G or elastase to normal bronchoalveolar lavage (BAL) fluid caused a dose dependent degradation of endogenous native SP-A. Cathepsin G and elastase were present in the BAL fluid from many patients with CF. Simple incubation of protease positive BAL fluid from patients with CF caused a time dependent degradation of added SP-A or, where present, endogenous SP-A. The degradation of SP-A by protease(s) in BAL fluid of patients with CF was abrogated by diisopropylfluorophosphate and monocyte/neutrophil elastase inhibitor. Conclusions: The findings strongly suggest that the neutrophil serine proteases cathepsin G and/or elastase and/or proteinase-3 contribute to degradation of SP-A and thereby diminish innate pulmonary antimicrobial defence.

A Novel Notch Protein, N2N, Targeted by Neutrophil Elastase and Implicated in Hereditary Neutropenia

Abstract: Mutations in ELA2, encoding the human serine protease neutrophil elastase, cause cyclic and severe congenital neutropenia, and recent evidence indicates that the mutations alter the membrane trafficking of neutrophil elastase. These disorders feature impaired bone marrow production of neutrophils along with excess monocytes—terminally differentiated lineages corresponding to the two alternative fates of myeloid progenitor cells. We utilized a modified yeast two-hybrid system and identified a new, widely expressed gene, N2N, whose product is homologous to Notch2, that interacts with neutrophil elastase. N2N is a 36-kDa protein distributed throughout the cell and secreted. Its amino-terminal sequence consists of several EGF repeats identical to those of the extracellular region of Notch2, and its carboxyl terminus contains a unique 24-residue domain required for interaction with neutrophil elastase. Neutrophil elastase cleaves N2N within EGF repeats in vitro and in living cells, but the C-terminal domain retards proteolysis. In vitro, N2N represses transcriptional activities of Notch proteins. Disease-causing mutations of neutrophil elastase disrupt the interaction with N2N, impair proteolysis of N2N and Notch2, and interfere with Notch2 signaling, suggesting defective proteolysis of an inhibitory form of Notch as an explanation for the alternate switching of cell fates characteristic of hereditary neutropenia.

Neutrophil elastase induces IL-8 synthesis by lung epithelial cells via the mitogen-activated protein kinase pathway

Abstract: The sequestration of neutrophils in the lung and the release of proinflammatory mediators, including neutrophil elastase, are responsible for sepsis-induced micro-vascular permeability and alveolar epithelial cell damage. To assess the underlying mechanism, human neutrophil elastase (0.01–0.5 µg/ml) was added to cultured A549 epithelial cells in the presence or absence of inhibitors. IL-8 was analyzed by ELISA or by RT-PCR to measure the IL-8 synthesis capacity. Mitogen-activated protein kinase (MAPK) activity was detected by Western blot analysis. Neutrophil elastase dose-dependently increased IL-8 release from cultured A549 epithelial cells. Pretreatment with a specific elastase inhibitor, elastase inhibitor II (at 0.5, 5, and 50 µg/ml), dose-dependently inhibited neutrophil elastase-induced IL-8 release. The activities of MAPK, p38, and extracellular signal-regulated kinase (ERK) were upregulated by neutrophil elastase. Nuclear transcriptional factor-kappa B (NF-kB) and activator protein 1 (AP-1) were also activated. These responses were significantly inhibited by elastase inhibitor II. A specific inhibitor of p38 MAPK (SB203580) and an NF-kB inhibitor (pyrrolidine dithiocarbamate), but not an ERK inhibitor (PD 98059), significantly inhibited neutrophil elastase-induced IL-8 release and mRNA expression. The specific tyrosine kinase inhibitor, genistein, and the protein kinase C (PKC) inhibitor, Ro 31-8220, also inhibited IL-8 release and mRNA expression as well as p38 and NF-kB activation. There was no significant effect by the protein kinase A inhibitor, H-89, on neutrophil elastase-induced IL-8 synthesis or p38 MAPK activation. Our results indicate that neutrophil elastase activates p38 MAPK which upregulates NF-kB and AP-1 activities, thus inducing IL-8 mRNA expression and protein synthesis. Tyrosine kinase and PKC are implicated in neutrophil elastase activation of the MAPK pathway.

Human leukocyte elastase and cathepsin G are specific inhibitors of C5a-dependent neutrophil enzyme release and chemotaxis.

Abstract: Circulating human neutrophils from patients with severe inflammatory disorders such as erysipelas and sepsis are specifically desensitized to complement factor C5a stimulation but not to stimulation with other stimuli like N-formyl-methionyl-leucyl-phenylalanine (FMLP), interleukin-8 (IL-8), leukotriene B4 (LTB4), or platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) In this study, we raised the question whether factors released from polymorphonuclear leukocytes (PMNs) can specifically down-regulate C5a-dependent neutrophil functions When neutrophils were preincubated with either neutrophil lysates or neutrophil degranulation supernatants, a complete inhibition of C5a-stimulated β-glucuronidase release and chemotaxis could be observed, whereas FMLP-, IL-8-, LTB4- or PAF-dependent functions were not affected Serine protease inhibitors like phenylmethylsulfonyl fluoride, antileukoprotease, or elafin abolished this effect High-performance liquid chromatography of neutrophil degranulation supernatants revealed pronounced inhibition of C5a-dependent neutrophil functions in fractions exerting elastase or cathepsin G activity, but not in fractions exerting proteinase 3 activity Using purified human leukocyte elastase (HLE), C5a responses like intracellular calcium influx, β-glucuronidase release, and chemotaxis were also specifically inhibited Our experiments show that the release of HLE or cathepsin G from neutrophils specifically down-regulates the responsiveness of neutrophils to C5a Elastase and cathepsin G may therefore play an important role in the down-regulation of acute inflammation

NF-κB p50 facilitates neutrophil accumulation during LPS-induced pulmonary inflammation

Abstract: Background: Transcription factors have distinct functions in regulating immune responses. During Escherichia coli pneumonia, deficiency of NF-κB p50 increases gene expression and neutrophil recruitment, suggesting that p50 normally limits these innate immune responses. p50deficient mice were used to determine how p50 regulates responses to a simpler, non-viable bacterial stimulus in the lungs, E. coli lipopolysaccharide (LPS). Results: In contrast to previous results with living E. coli, neutrophil accumulation elicited by E. coli LPS in the lungs was decreased by p50 deficiency, to approximately 30% of wild type levels. Heatkilled E. coli induced neutrophil accumulation which was not decreased by p50 deficiency, demonstrating that bacterial growth and metabolism were not responsible for the different responses to bacteria and LPS. p50 deficiency increased the LPS-induced expression of κBregulated genes essential to neutrophil recruitment, including KC, MIP-2, ICAM-1, and TNF-α suggesting that p50 normally limited this gene expression and that decreased neutrophil recruitment did not result from insufficient expression of these genes. Neutrophils were responsive to the chemokine KC in the peripheral blood of p50-deficient mice with or without LPSinduced pulmonary inflammation. Interleukin-6 (IL-6), previously demonstrated to decrease LPSinduced neutrophil recruitment in the lungs, was increased by p50 deficiency, but LPS-induced neutrophil recruitment was decreased by p50 deficiency even in IL-6 deficient mice. Conclusion: p50 makes essential contributions to neutrophil accumulation elicited by LPS in the lungs. This p50-dependent pathway for neutrophil accumulation can be overcome by bacterial products other than LPS and does not require IL-6.

Neutrophil activation in vitro

Abstract: Kinetics of spontaneous and induced reactions of Nitroblue tetrazolium (NBT) restoration by neutrophils was investigated in vitro. The index of activated neutrophils (IAN), commonly characterizing cell activation degree depending on the reaction time (t), was described by the equation: IAN = [NBT0](1-exp[-k1t]) + k0t; where (NBT0) is intracellular concentration of NBT, which reflects membrane permeability, k1 and k0--speed constants of the first and zero order reactions, characterizing, respectively, stimulation and spontaneous activities of enzymatic systems involved in NBT restoration. We detected the ability of heparin (2.0-7.5 mkg/ml) or chondrotin sulphate (0.25-2.5 mkg/ml) to activate neutrophils and presumably to maintain in vivo their spontaneous activity. The values of kinetics equation parameters enable us to speculate about relative hyper-, hypo- and normal function of neutrophils, related to the functional state of the whole organism. The value of [NBT0] changes from 8 to 103 conventional units, and a high correlation exists between this index and the reaction speed constants, differing in stimulated and spontaneously activated cells. The latter are represented by neutrophils only, which, as shown before, produce active forms of oxygen, used, probably, not for phagocytosis needs. The share of such cells makes approximately 30-40% of the neutrophil recirculating pool, and positively correlates with [NBT0], (r = 0.58; P < 0.05). This is determined by a very high membrane permeability of this neutrophil subpopulation. The detected different spontaneous and stimulated ability, various membrane permeability and enzyme system activity confirm the neutrophil functional unequality, which defines their activation peculiarity.

Modulation of LPS-induced suppression of neutrophil apoptosis by antibacterial cathelicidin peptide CAP11

Abstract: Peptide antibiotics possess potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. We previously revealed that antibacterial cathelicidin CAP11 (cationic antibacterial polypeptide of 11 kDa) exhibits protective actions against endotoxin shock model. During Gram-negative bacterial sepsis, lipopolysaccharide (LPS) activates neutrophils and their apoptosis is suppressed. Prolonged presence of activated neutrophils causes uncontrolled release of toxic metabolites, leading to the systemic tissue injury. In this study, we investigate the action of CAP11 on LPS-induced suppression of neutrophil apoptosis using human neutrophils.LPS suppressed neutrophil apoptosis, accompanied with the activation of NF-κB, phosphorylation of extracellular signal-related protein kinase, expression of Bcl-XL and inhibition of caspase 3 activation. Interestingly, CAP11 reversed the actions of LPS to trigger these changes, and induced neutrophil apoptosis. Furthermore, LPS activated monocytes to produce anti-apoptotic cytokines (IL-1β, TNF-α and IL-8) and suppressed neutrophil apoptosis. Importantly, CAP11 inhibited the cytokine production from LPS-stimulated monocytes, and induced neutrophil apoptosis. Finally, CAP11 strongly suppressed the LPS-binding to neutrophils and monocytes. These observations indicate that CAP11 can block the LPS-induced prolongation of neutrophil survival via the suppression of anti-apoptotic signaling in neutrophils and anti-apoptotic cytokine production from monocytes by inhibiting the binding of LPS to target cells.