The formation and function of the neutrophil phagosome

Neutrophils are the most abundant circulating leukocyte and are crucial to the initial innate immune response to infection. One of their key pathogen-eliminating mechanisms is phagocytosis, the process of particle engulfment into a vacuole-like structure called the phagosome. The antimicrobial activity of the phagocytic process results from a collaboration of multiple systems and mechanisms within this organelle, where a complex interplay of ion fluxes, pH, reactive oxygen species, and antimicrobial proteins creates a dynamic antimicrobial environment. This complexity, combined with the difficulties of studying neutrophils ex vivo, has led to gaps in our knowledge of how the neutrophil phagosome optimizes pathogen killing. In particular, controversy has arisen regarding the relative contribution and integration of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived antimicrobial agents and granule-delivered antimicrobial proteins. Clinical syndromes arising from dysfunction in these systems in humans allow useful insight into these mechanisms, but their redundancy and synergy add to the complexity. In this article, we review the current knowledge regarding the formation and function of the neutrophil phagosome, examine new insights into the phagosomal environment that have been permitted by technological advances in recent years, and discuss aspects of the phagocytic process that are still under debate.     

细菌性脑膜炎的氧化应激与抗氧化治疗

细菌性脑膜炎(BM)确切的发病机制尚不完全清楚，氧化应激所致神经损伤是BM的病理机制之一。大量研究证实氧化应激可以引起大脑皮层神经元的坏死和海马神经元的凋亡，而这些神经损伤均与脑膜炎后神经系统后遗症密切相关。研究发现，外源性给予维生素类、化学合成类、酶类等抗氧化剂对细菌性脑膜炎的神经损伤有一定的保护作用。     

T lymphocytes in acute bacterial infection: increased prevalence of CD11b(+) cells in the peripheral blood and recruitment to the infected site

T-cell activation, particularly of CD8(+) cells, is invariably associated with viral infections. We now provide evidence for the activation of T cells in patients with localized bacterial soft tissue infections. During acute disease we detected in the peripheral blood of these patients, small though conspicuous populations of CD4(+) CD28(+) CD11b(+) and CD8(+) CD28(+) CD11b(+) cells, indicative of an expansion of effector T cells. Moreover, we identified CD4(+) and CD8(+) cells at the infected site, in addition to highly activated polymorphonuclear neutrophils (PMN). In keeping with their role as first-line defence, PMN were preponderant, but T cells amounted to 20% of the infiltrated cells. The majority of the infiltrated T cells expressed CXCR6, a homing receptor for non-lymphoid tissue. The infiltrated T cells produced interferon-gamma (IFN-gamma), while the peripheral blood cells obtained at the same time did not. In conclusion, in response to localized bacterial infections, T cells are activated and recruited to the infected site. We propose that these T cells, e.g. by producing IFN-gamma, enhance the efficiency of the infiltrated phagocytic cells, particularly of the PMN, thereby supporting the local host defence.     

Chronic bacterial pulmonary infections in advanced cystic fibrosis differently affect the level of sputum neutrophil elastase,IL‐8 and IL‐6

Advanced cystic fibrosis (CF) lung disease is commonly characterized by a chronic Pseudomonas aeruginosa infection and destructive inflammation caused by neutrophils. However, the lack of convincing evidence from most informative biomarkers of severe lung dysfunction (SLD‐CF) has hampered the formulation of a conclusive, targeted diagnosis of CF. The aim of this study was to determine whether SLD‐CF is related to the high concentration of sputum inflammatory mediators and the presence of biofilm‐forming bacterial strains. Forty‐one patients with advanced CF lung disease were studied. The severity of pulmonary dysfunction was defined by forced expiratory volume in 1 second (FEV1) < 40%. C‐reactive protein (CRP) and NLR (neutrophil–lymphocyte ratio) were examined as representative blood‐based markers of inflammation. Expectorated sputum was collected and analysed for cytokines and neutrophil‐derived defence proteins. Isolated sputum bacteria were identified and their biofilm‐forming capacity was determined. There was no association between FEV1% and total number of sputum bacteria. However, in the high biofilm‐forming group the median FEV1 was < 40%. Importantly, high density of sputum bacteria was associated with increased concentrations of neutrophil elastase and interleukin (IL)‐8 and low concentrations of IL‐6 and IL‐10. The low concentration of sputum IL‐6 is unique for CF and distinct from that observed in other chronic pulmonary inflammatory diseases. These findings strongly suggest that expectorated sputum is an informative source of pulmonary biomarkers representative for advanced CF and may replace more invasive bronchoalveolar lavage analysis to monitor the disease. We recommend to use of the following inflammatory biomarkers: blood CRP, NLR and sputum elastase, IL‐6, IL‐8 and IL‐10.     

Granulocyte function in the airways of allergen-challenged pigs: effects of inhaled and systemic budesonide

Backgroumd Late airways obstruction and eosinophil infiltration after allergen challenge are often seen in human asthma and animal models of allergy. This inflammatory reaction, which may be a link between acute and chronic asthma, is blocked by glucocorticoid pretreatment. However, the role of eosinophils in late airways obstruction and the primary site of action of glucocorticoids, i.e. locally or systemically, have not been fully determined. Objectives This study was initiated to find out the role of eosinophils and neutrophils in allergen-induced late airways obstruction in the pig. The effect of pretreatment with budesonide (BUD) given locally or systemically on cellular responses seen within 8 h after allergen challenge was also studied. Methods Twenty-five minipigs were actively sensitized with Ascaris suum antigen and challenged under anaesthesia with antigen in the lower airways. Pigs were given BUD as an aerosol (l0μg/kg) or an intravenous infusion (5μg/kg) 1 h before allergen challenge. In one group, high doses of BUD (50μg/kg) were infused twice with a 3-h interval before allergen challenge. As a positive control, one group was given the BUD vehicle as an infusion and as a negative control, one group not treated with BUD was given the irrelevant antigen ovalbumin. Eosinophils and neutrophils in lung tissue specimens were detected and levels of eosinophil peroxidase (EPO) and myeloperoxidase (MPO) in bronchoalveolar lavage (BAL) fluid were measured using specific antibodies against porcine EPO and MPO. Results The number of eosinophils in lung tissue and BAL fluid and the level of EPO in BAL fluid were significantly increased 8 h after A. suum challenge in pigs not treated with BUD. With regard to possible recruitment and activation of neutrophils the only significant finding was an increase in the number of cells in BAL fluid. The eosinophil numbers and the level of EPO in BAL fluid were shown to be decreased by all BUD treatments in all the compartments studied compared to the positive control. However, the number of eosinophils in lung tissue and EPO levels in BAL fluid did not correlate with the magnitude of the late airways obstruction. Conclusion Although eosinophils are present in the bronchial wall and lumen and are apparently activated, a causative relationship between this granulocyte and the late bronchial obstruction could not be established in this model.     

Expression of the CXCR6 on polymorphonuclear neutrophils in pancreatic carcinoma and in acute, localized bacterial infections

The chemokine receptor CXCR6 has been described on lymphoid cells and is thought to participate in the homing of activated T-cells to non-lymphoid tissue. We now provide evidence that the chemokine receptor CXCR6 is also expressed by activated polymorphonuclear neutrophils (PMN) in vivo: Examination of biopsies derived from patients with pancreatic carcinoma by confocal laser scan microscopy revealed a massive infiltration of PMN that expressed CXCR6, while PMN of the peripheral blood of these patients did not. To answer the question whether CXCR6 expression is a property of infiltrated and activated PMN, leucocytes were collected from patients with localized soft tissue infections in the course of the wound debridement. By cytofluorometry, the majority of these cells were identified as PMN. Up to 50% of these PMN were also positive for CXCR6. Again, PMN from the peripheral blood of these patients were nearly negative for CXCR6, as were PMN of healthy donors. In a series of in vitro experiments, up-regulation of CXCR6 on PMN of healthy donors by a variety of cytokines was tested. So far, a minor, although reproducible, effect of tumour necrosis factor (TNFalpha) was seen: brief exposure with low-dose TNFalpha induced expression of CXCR6 on the surface of PMN. Furthermore, we could show an increased migration of PMN induced by the axis CXCL16 and CXCR6. In summary, our data provide evidence that CXCR6 is not constitutively expressed on PMN, but is up-regulated under inflammatory conditions and mediates migration of CXCR6-positive PMN.     

Neutrophil recognition of bacterial DNA and Toll-like receptor 9-dependent and -independent regulation of neutrophil function

Neutrophils are essential for host defense and detect the presence of invading microorganisms through recognition of pathogen-associated molecular patterns. Among these receptors are Toll-like receptors (TLRs). Neutrophils express all known TLRs except for TLR3. TLR9, localized intracellularly, is to date the best characterized sensor for bacterial DNA, containing short sequences of unmethylated CpG motifs, though TLR9-independent intracellular DNA recognition mechanism(s) may also exist. Bacterial DNA has profound impact on neutrophil functions; it promotes neutrophil trafficking in vivo, induces chemokine expression, regulates expression of adhesion molecules, enhances phagocyte activity, and rescues neutrophils from constitutive apoptosis. TLR9 stimulation results in alterations in cellular redox balance, peroxynitrite formation, activation of the mitogen-activated protein kinase, PI3-kinase, and Jun N-terminal kinase pathways and/or nuclear factor κB and AP-1. These features identify an important role for bacterial DNA and TLR9 signaling in the regulation of neutrophil functions that are critical for optimal expression as well as for resolution of the inflammatory response.     

A simple rapid technique to measure neutrophil or serum bactericidal activity

A rapid and simple technique to measure both serum and neutrophil bactericidal activity is described. It is based on microtitre equipment and avoids the cumbersome and time consuming serial dilution and plate counting of conventional experiments. A constant concentration of neutrophils and/or serum is incubated with a series of dilutions of bacterial suspensions. Bacterial survival is estimated at various times from their ability to form colonies on agar and the results conveniently expressed as the largest bacterial population that can be eliminated by either neutrophils or serum alone. The technique is as simple and quick as a standard microtitre MBC test commonly used for antibiotics and scoring of the results takes only a few seconds per sample. Data are presented showing the close correlation between results obtained using this technique and those from conventional tests of neutrophil bactericidal activity and phagocytosis-associated chemiluminescence.     

Rapid microassays of phagocytosis, bacterial killing, superoxide and hydrogen peroxide production by human neutrophils in vitro

Simple, rapid microassays for simultaneous measurement of phagocytosis, bacterial killing, superoxide and hydrogen peroxide production by human neutrophils in vitro are described. All assays employ 96-well flat bottom tissue culture plates which were incubated on a microtitre plate shaker at 37 degrees C. The separate evaluation of ingestion and intracellular killing of E. coli and S. aureus was based on the incorporation of [3H]uridine into viable extracellular bacteria. There was good correlation between plate counts of viable bacteria and amount of radiolabel incorporation. Phagocytosis and killing can be measured in a maximum of 100 microliter reaction mixture, requiring only 2.5 X 10(5) neutrophils per test and the assay is complete within 60 min. Assay of superoxide production by stimulated neutrophils was based on superoxide-dependent reduction of ferricytochrome c as measured spectrophotometrically at 550 nm in wells of tissue culture plates containing 150 microliter of reaction mixture. The assay requires only 1.25 X 10(5) neutrophils per test and is complete within 50 min. Quantitation of hydrogen peroxide was based on horseradish peroxidase-dependent oxidation of phenol red. The technique is as for superoxide detection except that the reaction must be terminated by the addition of 1 M NaOH at the desired time intervals. None of the assays require sampling during the incubation period. The principal advantages of the described techniques are increased simplicity and speed, requirement of low numbers of neutrophils and applicability to analysis of large number of samples in parallel.     

Anti-inflammatory and immunomodulatory effects of Spirulina platensis in comparison to Dunaliella salina in acetic acid-induced rat experimental colitis

Abstract

Context: Spirulina platensis (SP) is used as a source of protein and vitamin supplement in humans without any significant side-effects. Dunaliella salina (DS) is also regarded as one of the richest natural producers of carotenoid, thus used as a source of antioxidants to protect cells from oxidative damage.

Objective: The aim of the present study is to compare the ameliorative effect of Spirulina and Dunaliella in experimental colitis.

Materials and methods: Spirulina and Dunaliella were investigated at the same dose of 500?mg/kg body weight for their modulatory effect against acetic-acid induced ulcerative colitis (UC) in rats. The colonic lesion was analyzed by examining macroscopic damage, bloody diarrhea scores, colon weight/length and change in body weight of tested rats. Colon lipid peroxidation and oxidative stress markers were examined by evaluating malondialdehyde (MDA), protein carbonyl (PCO), catalase (CAT), reduced glutathione (GSH) and superoxide dismutase (SOD). Colon inflammatory markers; myeloperoxidase (MPO) and prostaglandin (PGE₂) as well as proinflammatory cytokines; tumor necrosis factor (TNF-α) and interleukins (IL-1β, IL-6) were also studied.

Results: The colonic mucosal injury, biochemical and histopathologic results suggest that both SP and DS exhibit significant modulatory effect on acetic acid-induced colitis in rats, which may be due to a significant increase of antioxidant enzymes activity and significant inhibition of lipid peroxidation and inflammation markers.

Discussion: Results showed that in comparison to Sulfasalazine, SP exhibited better therapeutic and safety profile than DS against acetic acid-induced UC.

Conclusion: This study suggests potential benefits of SP and DS in an experimental model of colitis.     

Regulatory Role of Nitric Oxide in Neutrophil Apoptosis

Apoptosis of blood neutrophils from healthy donors was studied under conditions of cell culturing with different concentrations of H₂O₂, selective NO synthase inhibitor, and inductor of NO synthesis (L-arginine). In vitro incubation of neutrophilic leukocytes with 5 mM H₂O₂ led to activation of the apoptotic program in neutrophils, which was seen from increased content of Bax protein in the cells and increased number of apoptotic cells in the culture. Increased content of annexin-positive cells after incubation of neutrophil culture with NO synthase inhibitor suggests involvement of NO in the regulation of neutrophil apoptosis under conditions of oxidative stress, while L-arginine prevented H₂O₂-induced programmed cell death. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 12, pp. 646–650, December, 2008     

消化道应用微磁囊定位系统抗地磁干扰的补偿方法

本研究介绍了一种应用于消化道微磁囊磁定位中的补偿地磁场干扰的方法.该方法通过设置补偿值来标记地磁场的干扰水平,并且用测量值减去补偿值来达到补偿干扰的目的.通过实验表明,该方法可以准确地确定定位背心所处的地磁场干扰水平;三维跟踪轨迹表明,该方法很大程度提高了微磁囊在消化道中定位的准确度.     

How human neutrophils kill and degrade microbes: an integrated view

Summary:  Neutrophils constitute the dominant cell in the circulation that mediates the earliest innate immune human responses to infection. The morbidity and mortality from infection rise dramatically in patients with quantitative or qualitative neutrophil defects, providing clinical confirmation of the important role of normal neutrophils for human health. Neutrophil-dependent anti-microbial activity against ingested microbes represents the collaboration of multiple agents, including those prefabricated during granulocyte development in the bone marrow and those generated de novo following neutrophil activation. Furthermore, neutrophils cooperate with extracellular agents as well as other immune cells to optimally kill and degrade invading microbes. This brief review focuses attention on two examples of the integrated nature of neutrophil-mediated anti-microbial action within the phagosome. The importance and complexity of myeloperoxidase-mediated events illustrate a collaboration of anti-microbial responses that are endogenous to the neutrophil, whereas the synergy between the phagocyte NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and plasma-derived group IIA phospholipase A₂ exemplifies the collective effects of the neutrophil with an exogenous factor to achieve degradation of ingested staphylococci.     

A study of 25 patients with chronic granulomatous disease: A new classification by correlating respiratory burst,cytochrome b,and flavoprotein

Twenty-five patients suffering from chronic granulomatous disease (CGD) and their families were investigated. Defects in the superoxide generating system were characterized at the level of the heme-containing cytochrome b and of the FAD-containing flavoprotein, both localized in the plasma membrane of granulocytes. It was confirmed that in most of the typical cases (18 of 22), the complete inability of superoxide generation was associated with the absence of detectable cytochrome b. Mothers but not fathers of such male patients were characterized by a diminished content of cytochrome b, confirming that the affected gene is localized on the X chromosome. In contrast, the granulocytes of four other typical patients (two female and two male) contained normal amounts of cytochrome b, whereas oxidative activity was absent. Since no abnormality of oxidative activity as well as of cytochrome b was found in granulocytes of the mothers and fathers of these patients, an autosomal recessive mode of inheritance of the disease is probable. The flavoprotein deficiency found in the granulocytes of four male patients was always associated with an absence of detectable cytochrome b. This could indicate a structural relationship between flavoprotein and cytochrome b (e.g., a flavocytochrome). Three further patients with mild X-linked CGD contrasted with the patients with severe or classic X-linked disease; the oxidative activity of their phagocytes was diminished but not absent, and the cytochrome b present, albeit in small amounts.     

Cyclic AMP-elevating agents down-regulate the oxidative burst induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) in adherent neutrophils.

Human neutrophils, plated on fibronectin-precoated wells, were found to release large quantities of superoxide anion (O2-) in response to GM-CSF. O2- production was reduced by prostaglandin E2 (PGE2) and the phosphodiesterase type IV (PDE IV) inhibitor RO 20-1724. Both agents are known to increase intracellular cyclic AMP (cAMP) levels by inducing its production (PGE2) or blocking its catabolism (RO 20-1724). When added in combination, PGE2 and RO 20-1724 had a marked synergistic inhibitory effect, which was reproduced by replacing PGE2 with a direct activator of adenylate cyclase, i.e. forskolin (FK). Moreover, the neutrophil response to GM-CSF was inhibited by a membrane-permeable analogue of cAMP in a dose-dependent manner. As GM-CSF and PGE2 are known to be generated at tissue sites of inflammation, the results suggest the existence of a PGE2-dependent regulatory pathway potentially capable of controlling the neutrophil response to GM-CSF, in turn limiting the risk of local oxidative tissue injury. Moreover, owing to its susceptibility to amplification by RO 20-1724, the PGE2-dependent pathway and in particular PDE-IV may represent a pharmacological target to reduce the generation of histotoxic oxidants by GM-CSF-responding neutrophils.     

The streptomycin mouse model for Salmonella diarrhea: functional analysis of the microbiota, the pathogen's virulence factors, and the host's mucosal immune response

The mammalian intestine is colonized by a dense microbial community, the microbiota. Homeostatic and symbiotic interactions facilitate the peaceful co-existence between the microbiota and the host, and inhibit colonization by most incoming pathogens ('colonization resistance'). However, if pathogenic intruders overcome colonization resistance, a fierce, innate inflammatory defense can be mounted within hours, the adaptive arm of the immune system is initiated, and the pathogen is fought back. The molecular nature of the homeostatic interactions, the pathogen's ability to overcome colonization resistance, and the triggering of native and adaptive mucosal immune responses are still poorly understood. To study these mechanisms, the streptomycin mouse model for Salmonella diarrhea is of great value. Here, we review how S. Typhimurium triggers mucosal immune responses by active (virulence factor elicited) and passive (MyD88-dependent) mechanisms and introduce the S. Typhimurium mutants available for focusing on either response. Interestingly, mucosal defense turns out to be a double-edged sword, limiting pathogen burdens in the gut tissue but enhancing pathogen growth in the gut lumen. This model allows not only studying the molecular pathogenesis of Salmonella diarrhea but also is ideally suited for analyzing innate defenses, microbe handling by mucosal phagocytes, adaptive secretory immunoglobulin A responses, probing microbiota function, and homeostatic microbiota-host interactions. Finally, we discuss the general need for defined assay conditions when using animal models for enteric infections and the central importance of littermate controls.