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Human Neutrophils (human + neutrophil)
Terms modified by Human Neutrophils Selected AbstractsFrom pro defensins to defensins: synthesis and characterization of human neutrophil pro ,-defensin-1 and its mature domainCHEMICAL BIOLOGY & DRUG DESIGN, Issue 2 2003Z. Wu Abstract: Human neutrophil ,-defensins (HNPs) are small, cationic, Cys-rich antimicrobial proteins that play important roles in innate immunity against infectious microbes such as bacteria, fungi and enveloped viruses. Synthesized as inactive precursors in vivo (pre-proHNPs), HNPs are activated through proteolytic removal of the inhibitory pro-peptide required for subcellular sorting and correct folding. We seek to understand the molecular basis for the recognition between the 45-residue pro-peptide and the C-terminal functional domain. Here we described, total chemical synthesis of the 75-residue human neutrophil pro ,-defensin-1 (proHNP1) via native chemical ligation. After oxidative folding, proHNP1 is cleaved by cyanogen bromide at the Met45,Ala46 peptide bond to release the mature form. The native disulfide connectivity in HNP1, i.e. Cys1,Cys6, Cys2,Cys4 and Cys3,Cys5, is verified by mass mapping of peptide fragments generated by proteolytic digestion and Edman degradation. Fluorescence spectroscopy studies and antimicrobial activity assays further support that synthetic proHNP1 and HNP1 are correctly folded. While largely unstructured in aqueous solution, the pro-peptide binds to HNP1 intermolecularly with an apparent Kd value of 6.2 ,m at pH 7.4, confirming the mode of intramolecular inactivation of human ,-defensin precursors. [source] Neutrophils display biphasic relationship between migration and substrate stiffnessCYTOSKELETON, Issue 6 2009Kimberly M. Stroka Abstract Neutrophils are one type of migrating cell in the body's innate immune system and are the first line of defense against inflammation or infection. While extensive work exists on the effect of adhesive proteins on neutrophil motility, little is known about how neutrophil motility is affected by the mechanical properties of their physical environment. This study investigated the effects of substrate stiffness on the morphology, random motility coefficient, track speed (v), spreading area, and distribution of turning angles of neutrophils during chemokinesis. Human neutrophils were plated onto polyacrylamide gels of varying stiffness, ranging from 3 to 13 kPa, and coated with the extracellular matrix protein fibronectin, and timelapse images were taken with phase contrast microscopy. Our results show a biphasic behavior between neutrophil motility and substrate stiffness, with the optimum stiffness for motility depending on the concentration of fibronectin on the surface of the gel. On 100 ,g/mL fibronectin, the optimum stiffness is 4 kPa (v = 6.9 ± 0.6 ,m/min) while on 10 ,g/mL fibronectin, the optimum stiffness increases to 7 kPa (v = 4.5 ± 2.0 ,m/min). This biphasic behavior most likely arises because neutrophils on soft gels are less adherent, preventing production of traction forces, while neutrophils on stiff gels adhere strongly, resulting in decreased migration. At intermediate stiffness, however, neutrophils can attain optimal motility as a function of extracellular matrix coating. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Microinjected neutrophils retain the ability to take up bacteriaJOURNAL OF ANATOMY, Issue 5 2002M. M. Bird It is now possible to microinject protein to probe specific biochemical pathways and/or cell functions in small cells such as human neutrophils (Bird et al. J.Anat.198, 2001). We have shown that these cells retain their ability to modify their F-actin cytoskeleton following the microinjection procedure. The principal task of neutrophils is to hunt and kill bacteria by responding to chemotactic gradients which cause them to extend actin rich pseudopodia in the direction of the highest concentration of these molecules. On reaching their target the neutrophils make tight contact with the bacteria and phagocytosis ensues. Here we address the question of whether or not the microinjected cells are still able to maintain their normal phagocytic activities. Human neutrophils maintained in culture for 20 mins were confronted with Staphylococcus aureus (1 × 104 cells/mL) for 5 min and then injected with rat IgG as an exogenous protein that also serves as a marker for injected cells. After 30 min the cells were fixed for fluorescence or confocal microscopy in 3.7% formaldehyde and permeabilised for 5 min (0.2% Triton X-100 in PBS). They were then incubated for 45 min in 2.5 µL FITC-anti rat IgG and 1 µL TRITC-phalloidin (to show the F-actin cytoskeleton), in 996.5 µL of PBS, washed 6 times in PBS and mounted on slides in 5 µL Mowiol containing a grain of antiquench. For TEM cells were fixed in 1.5% glutaraldehyde in cacodylate buffer for 3 min at room temperature and then washed in 0.2 m cacodylate buffer 6 times before incubation with 1 mm NiCl2 and SIGMA fast DAB peroxidase tablets for 30 min. The cells were postfixed in a 2% solution of osmium tetroxide for 30 min, dehydrated through a series of graded ethanols, and embedded and sectioned for TEM. By TEM the injected neutrophils were observed to have taken up bacteria into vacuoles of varying size. At the earliest stages of this process, prior to and immediately following the initial release of granular contents and the initiation of mechanisms to rapidly destroy bacteria, the bacteria fitted more tightly in the vacuoles than at later stages. Injected neutrophils commonly contained several bacteria; more than one bacterium was frequently located within a single vacuole of substantial size. Confocal laser microscopic observations confirmed that cells containing ingested bacteria also contained IgG. Thus injected cells not only survive the microinjection procedure but also retain their ability to take up bacteria and initiate the digestive process. [source] Free radical,scavenging activity and DNA damaging potential of auxins IAA and 2-methyl-IAA evaluated in human neutrophils by the alkaline comet assayJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 3 2010Branka Salopek-Sondi Auxins, of which indole-3-acetic acid (IAA) is the most widespread representative, are plant hormones. In addition to plants, IAA also naturally occurs in humans in micromolar concentrations. In the presence of peroxidase, indolic auxins are converted to cytotoxic oxidation products and have thus been proposed for use in gene-directed enzyme/prodrug tumor therapy. Since data on the genotoxicity of IAA and its derivatives are not consistent, here we investigate the early DNA damaging effects (2-h treatment) of the auxins, IAA, and 2-methyl-indole-3-acetic acid (2-Me-IAA) by the alkaline comet assay and compare them with their free radical,scavenging activity measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Human neutrophils are chosen as the test system since they possess inherent peroxidase activity. The results of the comet assay indicate an increase in DNA damage in a dose-dependent manner up to 1.00 mM of both auxins. Generally, IAA applied in the same concentration had greater potential to damage DNA in human neutrophils than did 2-Me-IAA. The genotoxicities of the two examined auxins are negatively correlated with their antioxidant activities, as measured by the DPPH assay; 2-Me-IAA showed a higher antioxidant capacity than did IAA. We assume that differences in the molecular structure of the tested auxins contributed to differences in their metabolism, in particular, with respect to interactions with peroxidases and other oxidative enzymes in neutrophils. However, the exact mechanisms have to be elucidated in future studies. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:165,173, 2010; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20323 [source] Calprotectin release from human neutrophils is induced by Porphyromonas gingivalis lipopolysaccharide via the CD-14,Toll-like receptor,nuclear factor ,B pathwayJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2003Jun-ichi Kido Objectives:, Calprotectin is a cytosolic protein with antibacterial action in leukocytes and its level increases in some inflammatory diseases, including periodontal diseases, rheumatoid arthritis and ulcerative colitis. Recently, we found that the lipopolysaccharide of Porphyromonas gingivalis (P-LPS) induced calprotectin release from human neutrophils. P-LPS, a major virulence factor of periodontal pathogens, is known to induce the production and release of inflammatory cytokines through CD14, Toll-like receptor (TLR) and nuclear factor ,B (NF-,B). In the present study, we investigated whether calprotectin release by P-LPS is induced via the CD14,TLR,NF-,B pathway and the cellular mechanism of calprotectin release in human neutrophils. Material and methods:, Human neutrophils were isolated from the peripheral blood of healthy donors and pre-incubated in medium containing antibodies against CD14, TLR2 and TLR4, or several inhibitors of NF-,B, microtubules and microfilaments, and then incubated with P-LPS. The calprotectin amount in the culture medium was determined using ELISA, and the nuclear extracts from cells were used for the examination of NF-,B binding activity using electrophoretic mobility shift assays. Results:, P-LPS increased calprotectin release from neutrophils and its induction was inhibited by anti-CD14 and anti-TLR2 antibodies, but not by two anti-TLR4 antibodies. NF-,B inhibitors suppressed P-LPS-induced NF-,B binding activity and calprotectin release. The inhibitors of microtubule and microfilament polymerization significantly decreased P-LPS-induced calprotectin release. Conclusion:, These results suggest that calprotectin release is induced by P-LPS via the CD14,TLR2,NF-,B signal pathway in human neutrophils and may be dependent on microtubule and microfilament systems. [source] Pyruvate Preserves Neutrophilic Superoxide Production in Acidic, High Glucose-Enriched Peritoneal Dialysis SolutionsARTIFICIAL ORGANS, Issue 3 2003Yi Tai Wu Abstract: Aim: To investigate effects of pyruvate (Py)-based peritoneal dialysis solutions (P-PDS) on neutrophilic superoxide (O2,) production against high glucose (HG) concentrations at acidic or physiologic pH value, and explore potential mechanisms. Methods: Human neutrophils were incubated with both dl -lactate (La, 40 mM)-based PDS (L-PDS) and equimolar P-PDS at various pH and HG levels, respectively. Hanks' balanced salt solution (HBSS) served as controls. O2, generation was determined by the reduction of ferricytochrome c. Results: Acidic pH and high La induced acute and substantial inhibitions of O2, production. HG in both PDS and HBSS resulted in a suppression of O2, in a dose-dependent manner. P-PDS generated near twofold O2, formation of L-PDS counterparts at various pH and HG levels. P-PDS with HG produced significantly more O2, than Py-free HBSS counterparts. Conclusions: Py in PDS effectively protected neutrophils from HG-induced inhibition of O2, production, even at a physiological pH. The Py cytoprotection may be associated with the preservation of carbohydrate metabolic pathways in addition to its alkalization. [source] Dapsone suppresses human neutrophil superoxide production and elastase release in a calcium-dependent mannerBRITISH JOURNAL OF DERMATOLOGY, Issue 5 2005T. Suda Summary Background, Dapsone (4,4,-diaminodiphenyl sulphone) is a powerful therapeutic tool in many skin diseases including neutrophilic dermatoses. The drug has an outstanding therapeutic efficacy against many skin diseases characterized by neutrophil-rich infiltrates; however, mechanisms of its action are poorly understood. Objectives, We investigated the effects of dapsone on respiratory and secretory functions of human neutrophils triggered by the chemotactic peptide N -formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), the physiological agonist C5a, and phorbol myristate acetate (PMA). Methods, Human neutrophils were isolated from venous blood obtained from healthy donors. We detected extracellular production of superoxide (O2,) by cytochrome C reduction assay, and intracellular production of O2, by flow cytometry. Neutrophil elastase release was measured by the cleavage of the specific elastase substrate N -methoxysuccinyl-Ala-Ala-Pro-Val- p -nitroanilide. Measurement of cytosolic free calcium concentration was performed using the calcium-reactive fluorescence probe, Fluo-3. Results, Dapsone suppressed intra- and extracellular production of O2, and elastase release triggered by fMLP and C5a, but not by PMA. Both fMLP and C5a signalled the above pathways by inducing calcium influx, but PMA functions bypassed calcium influx. Dapsone was capable of antagonizing the induction of calcium influx. Conclusions, These findings suggest that one mechanism of the anti-inflammatory action of dapsone is inhibition of calcium-dependent functions of neutrophils including release of tissue-damaging oxidants and proteases in the affected skin. [source] Respiratory syncytial virus and neutrophil activationCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2005E. L. Bataki Summary Respiratory syncytial virus infects almost all children by 2 years of age. Neutrophils are the predominant airway leucocytes in RSV bronchiolitis and they are activated in the presence of infection. However it is not clear whether RSV can directly signal to activate neutrophil cytotoxic function. To investigate this we have used a preparation of RSV washed using a new centrifugal diafiltration method to rapidly remove inflammatory molecules produced by the epithelial cells used to propagate the RSV stock. Human neutrophils were isolated from peripheral blood and activated with either the unwashed crude RSV preparations or the purified intact RSV. Neutrophils were also challenged with purified RSV G-glycoprotein. The effect of challenging human neutrophils with these preparations of intact RSV, or the RSV G-glycoprotein, was assessed by measuring the cell surface expression of CD11b and CD18b, the phagocytic oxidative burst, and intracellular release of calcium pools. Neutrophils challenged with the washed RSV exhibited significantly lower activation of surface marker expression (P < 0·001) and oxidative burst (P < 0·001) than those challenged with unwashed virus or with virus free supernatant. There was no increase in intracellular calcium release on exposure to the washed RSV. Purified G glycoprotein did not stimulate neutrophils, whilst the use of a blocking antibody to the F protein did not prevent unwashed RSV from activating cytotoxic responses. These results suggest that neutrophils have no innate signalling system that recognizes RSV but they are activated at sites of RSV infection as a result of the cytokines and inflammatory molecules released by virally infected cells. [source] Fibrinogen-CD11b/CD18 interaction activates the NF-,B pathway and delays apoptosis in human neutrophilsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2003Carolina Rubel Abstract The regulation of neutrophil half-life by members of the coagulation cascade is critical for the resolution of the inflammatory response. We have demonstrated that soluble fibrinogen (sFbg) delays human neutrophil (PMN) apoptosis through a mechanism that involves CD11b interactions, and phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase,1/2 (ERK1/2). Since NF-,B is a key element in the regulation of apoptotic mechanisms in several immune cells, we investigated whether NF-,B is involved in the control of PMN survival by sFbg. We showthat sFbg triggers inhibitor protein ,B (I,B-,) degradation and NF-,B activation. Furthermore, pharmacological inhibition of NF-,B abrogates sFbg effects on apoptosis. In addition, specific inhibition of MAPK ERK1/2 significantly reduces NF-,B translocation by sFbg, suggesting a relationship between ERK1/2 and NF-,B activation. Similar results are obtained when granulocytic-differentiated HL-60 cells are treated with sFbg, making this model highly attractive for integrin-induced gene expression studies. It can be concluded that NF-,B participates in the prevention of apoptosis induced by sFbg with the participation of MAPK ERK1/2. These results shed light on the molecular mechanisms that control human granulocyte apoptosis, and suggest that NF-,B regulation may be of benefit for the resolution of the inflammatory response. [source] Thymol analogues with antioxidant and L-type calcium current inhibitory activityDRUG DEVELOPMENT RESEARCH, Issue 4 2005Ai-Yu Shen Abstract Thymol is a natural product, which has antioxidant activity. 4-Morpholinomethyl-2-isopropyl-5-methylphenol (THMO), and 4-Pyrrolidinomethyl-2-isopropyl- 5-methylphenol (THPY) were synthesized by reacting thymol with formaldehyde and, respectively, morpholine or pyrrolidine. Since there is a relationship between the antioxidative status and incidence of human disease, anti-superoxidation, free radical scavenger activity, and anti-lipid peroxidation of the thymol analogues were determined by xanthine oxidase inhibition, cytochrome C system with superoxide anion releasing with formyl-Met-Leu-Phe (fMLP)/cytochalasin (CB) or phorbol myristate acetate (PMA) activating pathway in human neutrophils. All compounds studied had antioxidant activity. Mannich bases derived from thymol were generally found to be more potent compounds than thymol. THMO demonstrated the greatest antioxidant activity with IC50 values for xanthine oxidase inhibition and anti-lipid peroxidation being 21±2.78 and 61.29±5.83 µM, respectively. Moreover, since oxidative stress by free radical regulates the activity of L-type Ca2+ channel, the whole-cell configuration of the patch-clamp technique was used to investigate the effect of THMO upon ionic currents within NG108-15 cells. THMO (10 µM) suppressed the peak amplitude of L-type Ca2+ inward current (ICa,L), indicating that the antioxidative potential of the thymol analogues might be related to calcium current inhibition. The present studies suggest that THMO-dependent antioxidant and calcium ion current inhibition activity may be useful in treating free radical-related disorders. Drug Dev Res 64:195,202, 2005. © 2005 Wiley-Liss, Inc. [source] A 4-trifluoromethyl derivative of salicylate, triflusal, stimulates nitric oxide production by human neutrophils: role in platelet functionEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 9 2000De Miguel Background The thrombotic process is a multicellular phenomenon in which not only platelets but also neutrophils are involved. Recent in vitro studies performed in our laboratory have demonstrated that triflusal, a 4-trifluoromethyl derivative of salicylate, reduced platelet aggregation not only by inhibiting thromboxane A2 production but also by stimulating nitric oxide (NO) generation by neutrophils. The aim of the present study was to evaluate whether oral treatment of healthy volunteers with triflusal could modify the ability of their neutrophils to produce NO and to test the role of the NO released by neutrophils in the modulation of ADP-induced platelet aggregation and ,-granule secretion. Methods The study was performed in 12 healthy volunteers who were orally treated with triflusal (600 mg day,1) for 5 days. Flow cytometric detection of platelet surface expression of P-selectin was used as a measure of the ability of platelets to release the contents of their ,-granules. Results After treatment with triflusal, there was an increase in NO production by neutrophils and an increase in endothelial nitric oxide synthase (eNOS) protein expression in neutrophils. A potentiation of the inhibition of platelet aggregation by neutrophils was reversed by incubating neutrophils with both an l -arginine antagonist, NG -nitro- l -arginine methyl ester ( l -NAME) and an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5 tetramethylimidazoline 1-oxyl 3-oxide (C-PTIO). A slight decrease in P-selectin surface expression on platelets was found which was not modified by the presence of neutrophils and therefore by the neutrophil-derived NO. Exogenous NO released by sodium nitroprusside dose-dependently inhibited both ADP-stimulated ,-granule secretion and platelet aggregation. Therefore, platelet aggregation showed a greater sensitivity to be inhibited by exogenous NO than P-selectin expression. Conclusion Oral treatment of healthy volunteers with triflusal stimulated NO production and eNOS protein expression in their neutrophils. After triflusal treatment, the neutrophils demonstrated a higher ability to prevent ADP-induced platelet aggregation. However, the neutrophils and the endogenous NO generated by them failed to modify P-selectin expression in ADP-activated platelets. [source] IL-10 modulates cytokine gene transcription by protein synthesis-independent and dependent mechanisms in lipopolysaccharide-treated neutrophilsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2007Marzia Rossato Abstract We have recently reported that the ability of IL-10 to rapidly exert its anti-inflammatory effects on human neutrophils is dependent upon exposure of these cells to LPS for at least 3,4,h. Here, we demonstrate that, in neutrophils "preconditioned" by LPS, IL-10 primarily targets the transcription of TNF-,, CXCL8 and IL-1ra genes, as revealed by primary transcript real-time RT-PCR. We also show that IL-10-induced transcriptional repression of TNF-, and CXCL8 genes consists of two distinct phases: an early one, occurring rapidly and in a protein synthesis-independent manner, followed by a second phase, more delayed and dependent on protein synthesis. Interestingly, the protein synthesis dependence of the latter phase coincides with a reduced ability of IL-10 to induce STAT3 tyrosine phosphorylation. Importantly, inhibition of IL-10-induced STAT3 activation and IL-10-suppressive action by a prolonged exposure to cycloheximide (CHX) was observed to occur also in human monocytes and was caused by a defective IL-10-mediated activation of Jak1 and Tyk2 kinases. Taken together, our findings suggest that CHX interferes with the IL-10-mediated intracellular signaling pathway by interrupting events upstream of STAT3 activation. These data question the concept of the requirement of an IL-10-induced mediator as the unique mechanism to execute IL-10 anti-inflammatory program. [source] ADAM17 activity during human neutrophil activation and apoptosisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2006Bruce Walcheck Dr. Abstract Substrates of the metalloprotease ADAM17 (also known as TNF-, converting enzyme or TACE) undergo ectodomain shedding and include various inflammatory modulators. Though polymorphonuclear leukocytes contribute significantly to inflammation, direct analyses of ADAM17 on human neutrophils are very limited. In addition, the current understanding of the processes regulating ADAM17 activity primarily relate to its rapid activation. Therefore, to extend insights into the mechanisms of ADAM17 activity, we examined its surface expression and the shedding of its substrates during extended periods of neutrophil activation and apoptosis. Contrary to studies with immortalized hematopoietic cell lines, we report that surface expression of ADAM17 is maintained by human neutrophils activated with formyl peptides or by FcR/complement receptor-mediated phagocytosis. Interestingly, bacterial phagocytosis resulted in a significant increase in ADAM17 expression several hours after pathogen engulfment. We provide novel evidence that ADAM17 surface expression is also maintained during spontaneous and anti-Fas-induced neutrophil apoptosis. The well-validated ADAM17 substrates L-selectin and proTNF-, were shed efficiently by neutrophils under each of the conditions tested. Our data thus indicate prolonged ADAM17 expression during neutrophil effector functions. The implications of this may be a role by ADAM17 in both the induction and down-regulation of neutrophil activity. [source] Fibrinogen-CD11b/CD18 interaction activates the NF-,B pathway and delays apoptosis in human neutrophilsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2003Carolina Rubel Abstract The regulation of neutrophil half-life by members of the coagulation cascade is critical for the resolution of the inflammatory response. We have demonstrated that soluble fibrinogen (sFbg) delays human neutrophil (PMN) apoptosis through a mechanism that involves CD11b interactions, and phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase,1/2 (ERK1/2). Since NF-,B is a key element in the regulation of apoptotic mechanisms in several immune cells, we investigated whether NF-,B is involved in the control of PMN survival by sFbg. We showthat sFbg triggers inhibitor protein ,B (I,B-,) degradation and NF-,B activation. Furthermore, pharmacological inhibition of NF-,B abrogates sFbg effects on apoptosis. In addition, specific inhibition of MAPK ERK1/2 significantly reduces NF-,B translocation by sFbg, suggesting a relationship between ERK1/2 and NF-,B activation. Similar results are obtained when granulocytic-differentiated HL-60 cells are treated with sFbg, making this model highly attractive for integrin-induced gene expression studies. It can be concluded that NF-,B participates in the prevention of apoptosis induced by sFbg with the participation of MAPK ERK1/2. These results shed light on the molecular mechanisms that control human granulocyte apoptosis, and suggest that NF-,B regulation may be of benefit for the resolution of the inflammatory response. [source] Agonists of proteinase-activated receptor-2 affect transendothelial migration and apoptosis of human neutrophilsEXPERIMENTAL DERMATOLOGY, Issue 10 2007Victoria M. Shpacovitch Abstract:, Skin is the first barrier preventing microorganism invasion in host. Wounds destroy this defense barrier and, without an appropriate care, may lead to sepsis. Neutrophil activation and immigration plays an important role at the inflammatory stage of wound healing. Neutrophils are known to express proteinase-activated receptors (PARs), which can be activated by serine proteases, also by enzymes involved in wound healing. We previously reported that PAR2 agonists up-regulate cell adhesion molecule expression and cytokine production by human neutrophils. Here, we demonstrate that PAR2 agonists (serine proteases as well as synthetic peptides) reduce transendothelial migration of neutrophils and prolong their life in vitro. Synthetic PAR2 agonist also enhanced protective interferon (IFN),-induced Fc,RI expression at neutrophil cell surface. Of note, IFN, is a cytokine, which was used in clinical trials to reactivate human neutrophil functions during sepsis. Moreover, we observed a significant increase of PAR2 expression on cell surface of neutrophils from septic patients as compared with healthy volunteers. Together, our results indicate that PAR2 may be involved in the pathophysiology of neutrophil-endothelial interactions during wound healing or later during sepsis in humans, potentially by affecting neutrophil apoptosis, transendothelial migration and Fc, receptor-mediated phagocytosis. [source] Neutrophil gelatinase-associated lipocalin is a marker for dysregulated keratinocyte differentiation in human skinEXPERIMENTAL DERMATOLOGY, Issue 6 2002Lotus Mallbris Abstract: Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa protein initially isolated from the specific granules of human neutrophils. It is a member of the highly heterogeneous lipocalin protein family, which shares a common tertiary structure. Its synthesis is induced in gastrointestinal epithelium in association with inflammation and malignancy. To gain insight into its potential role in other epithelia we have investigated the expression of NGAL in human skin embryonic development, in normal adult skin, and in skin associated with inflammation and neoplastic transformation. In the present study we report that the embryonic expression of NGAL appears to be regulated in a spatio-temporal pattern. It was induced in the interfollicular epidermis at 20,24 weeks of gestational age but thereafter progressively receded towards the hair follicles. In normal adult skin, NGAL was detected solely in association with hair follicles. However, strong induction of NGAL in the epidermis was seen in a variety of skin disorders characterized by dysregulated epithelial differentiation such as psoriasis, pityriasis rubra and squamous cell carcinoma. In these tissues production of NGAL was confined to spatially distinct subpopulations of keratinocytes underlying areas of parakeratosis, whereas skin samples lacking parakeratotic epithelium such as lichen ruber planus, acute contact eczema and basal cell carcinoma were negative for NGAL. Consistent with being a marker for disturbed terminal differentiation, NGAL immunoreactivity showed an inverse pattern when compared with that of the differentiation marker filaggrin. The biologic functions of NGAL in epithelia are not fully known, although an immunomodulatory role in host defense has been proposed. In addition, the transient interfollicular NGAL expression during skin embryogenesis along with the induction of NGAL in adult parakeratotic epidermis suggests it play a role in epithelial differentiation pathways. [source] The identification of a phospholipase B precursor in human neutrophilsFEBS JOURNAL, Issue 1 2009Shengyuan Xu A phospholipase B (PLB) precursor was purified from normal human granulocytes using Sephadex G-75, Mono-S cation-exchange and hydroxyapatite columns. The molecular mass of the protein was estimated to be , 130 kDa by gel filtration and 22 and 42 kDa by SDS/PAGE. Tryptic peptide and sequence analyses by MALDI-TOF and tandem mass spectrometry (MS/MS) identified the protein as a FLJ22662 (Homo sapiens) gene product, a homologue of the amoeba Dictyostelium discoideum PLB. The native protein needed modifications to acquire deacylation activity against phospholipids including phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and lysophospholipids. Enzyme activity was associated with fragments derived from the 42 kDa fragment. The enzyme revealed a PLB nature by removing fatty acids from both the sn -1 and sn -2 positions of phospholipids. The enzyme is active at a broad pH range with an optimum of 7.4. Immunoblotting of neutrophil postnuclear supernatant using antibodies against the 42 kDa fragment detected a band at a molecular mass of 42 kDa, indicating a neutrophil origin of the novel PLB precursor. The existence of the PLB precursor in neutrophils and its enzymatic activity against phospholipids suggest a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation. [source] Role of Ca2+/calmodulin regulated signaling pathways in chemoattractant induced neutrophil effector functionsFEBS JOURNAL, Issue 18 2002Comparison with the role of phosphotidylinositol-3 kinase In human neutrophils, both changes in intracellular Ca2+ concentrations, [Ca2+]i, and activation of phosphatidylinositol-3 kinase (PtdIns3K) have been proposed to play a role in regulating cellular function induced by chemoattractants. In this study we have investigated the role of [Ca2+]i and its effector molecule calmodulin in human neutrophils. Increased [Ca2+]i alone was sufficient to induce phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2), p38 mitogen activated kinase (p38 MAPK), protein kinase B (PKB) and glycogen synthase kinase-3, (GSK-3,). Inhibition of calmodulin using a calmodulin antagonist N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), did not effect N -formyl-methionyl-leucyl-phenylalanine (fMLP) induced ERK, p38 MAPK or GSK-3, phosphorylation, but attenuated fMLP induced PKB phosphorylation. PCR analysis of human neutrophil cDNA demonstrated variable expression of members of the Ca2+/calmodulin-dependent kinase family. The roles of calmodulin and PtdIns3K in regulating neutrophil effector functions were further compared. Neutrophil migration was abrogated by inhibition of calmodulin, while no effect was observed when PtdIns3K was inhibited. In contrast, production of reactive oxygen species was sensitive to inhibition of both calmodulin and PtdIns3K. Finally, we demonstrated that chemoattractants are unable to modulate neutrophil survival, despite activation of PtdIns3K and elevation [Ca2+]i. Taken together, our data indicate critical roles for changes in [Ca2+]i and calmodulin activity in regulating neutrophil migration and respiratory burst and suggest that chemoattractant induced PKB phosphorylation may be mediated by a Ca2+/calmodulin sensitive pathway in human neutrophils. [source] Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophilsFEBS JOURNAL, Issue 15 2002Evidence for different arachidonate pools The goal of this study was to determine the effects of a putative specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), on arachidonic acid (AA) release and lipid mediator biosynthesis by ionophore-stimulated human neutrophils. Initial studies indicated that AACOCF3 at concentrations 0,10 µm did not affect AA release from neutrophils. In contrast, AACOCF3 potently inhibited leukotriene B4 formation by ionophore-stimulated neutrophils (IC50 , 2.5 µm). Likewise, AACOCF3 significantly inhibited the biosynthesis of platelet activating factor. In cell-free assay systems, 10 µm AACOCF3 inhibited 5-lipoxygenase and CoA-independent transacylase activities. [3H]AA labeling studies indicated thatthe specific activities of cell-associated AA mimicked that of leukotriene B4 and PtdCho/PtdIns, while the specific activities of AA released into the supernatant fluid closely mimicked that of PtdEtn. Taken together, these data argue for the existence of segregated pools of arachidonate in human neutrophils. One pool of AA is linked to lipid mediator biosynthesis while another pool provides free AA that is released from cells. Additionally, the data suggest that AACOCF3 is also an inhibitor of CoA-independent transacylase and 5-lipoxygenase. Thus, caution should be exercised in using AACOCF3 as an inhibitor of cytosolic phospholipase A2 in whole cell assays because of the complexity of AA metabolism. [source] Transcriptional profiling of the Candida albicans Ssk1p receiver domain point mutants and their virulenceFEMS YEAST RESEARCH, Issue 5 2008Veena Menon Abstract The Ssk1p response regulator of Candida albicans is required for oxidant adaptation, survival in human neutrophils, and virulence in a disseminated murine model of candidiasis. We have previously shown that the amino acid residues D556 and D513 of the Ssk1p receiver domain are critical to the Ssk1p in oxidant stress adaptation and morphogenesis. Herein, transcriptional profiling is used to explain the oxidant sensitivity and morphogenesis defect of two point mutants (D556N and D513K, respectively) compared with a WT strain. In the D556N mutant, during oxidative stress (5 mM H2O2), a downregulation of genes associated with redox homeostasis and oxidative stress occurred, which accounted for about 5% of all gene changes, including among others, SOD1 (superoxide dismutase), CAP1 (required for some types of oxidant stress), and three genes encoding glutathione biosynthesis proteins (GLR1, GSH1, and GSH2). Mutant D513K was not sensitive to peroxide but was impaired in its yeast $/to hyphal transition. We noted downregulation of genes associated with morphogenesis and cell elongation. Virulence of each mutant was also evaluated in a rat vaginitis model of candidiasis. Clearance of an SSK1 null and the D556N mutants from the vaginal canal was significantly greater than wild type or the D513K mutant, indicating that a change in a single amino acid of the Ssk1p alters the ability of this strain to colonize the rat vaginal mucosa. [source] Key role of proline-rich tyrosine kinase 2 in interleukin-8 (CXCL8/IL-8)-mediated human neutrophil chemotaxisIMMUNOLOGY, Issue 4 2004Vito Di Cioccio Summary The signalling pathways leading to CXCL8/IL-8-induced human neutrophil migration have not been fully characterized. The present study demonstrates that CXCL8 induces tyrosine phosphorylation as well as enzymatic activity of proline-rich tyrosine kinase 2 (Pyk2), a non-receptor protein tyrosine kinase (PTK), in human neutrophils. Induction of Pyk2 tyrosine phosphorylation by CXCL8 is regulated by Src PTK activation, whereas it is unaffected by phosphatidylinositol 3-kinase activation. Inhibition of Pyk2 activation by PP1, a Src PTK inhibitor, is paralleled by the inhibition of CXCL8-mediated neutrophil chemotaxis. Among CXCL8 receptors, Src protein tyrosine kinase activation selectively regulates CXCR1-mediated polymorphonuclear neutrophil (PMN) chemotaxis. Overexpression of PykM, the kinase-dead mutant of Pyk2, blocks CXCL8-induced chemotaxis of HL-60-derived PMN-like cells, thus pinpointing the key role of Pyk2 in CXCL8-induced chemotaxis. [source] Regulation of human neutrophil-mediated cartilage proteoglycan degradation by phosphatidylinositol-3-kinaseIMMUNOLOGY, Issue 1 2001C. S. T. Hii Summary The ability of neutrophils to degrade cartilage proteoglycan suggests that the neutrophils that accumulate in the joints of rheumatoid arthritis patients are mediators of tissue damage. The regulatory mechanisms which are relevant to the proteoglycan-degrading activity of neutrophils are poorly understood. Since phosphatidylinositol 3-kinase (PI3-K), protein kinase C (PKC), the extracellular signal-regulated protein kinase (ERK)1/ERK2 and cyclic adenosine monophosphate (cAMP) have been reported to regulate neutrophil respiratory burst and/or degranulation, a role for these signalling molecules in regulating proteoglycan degradation was investigated. Preincubation of human neutrophils with GF109203X (an inhibitor of PKC), PD98059 (an inhibitor of MEK, the upstream regulator of ERK1/ERK2) or with forskolin or dibutyryl cAMP, failed to suppress proteoglycan degradation of opsonized bovine cartilage. In contrast, preincubation of neutrophils with wortmannin or LY294002, specific inhibitors of PI3-K, inhibited proteoglycan degradation. Incubation of neutrophils with cartilage resulted in the activation of PI3-K in neutrophils, consistent with a role for PI3-K in proteoglycan degradation. Activation of PI3-K and proteoglycan degradation was enhanced by tumour necrosis factor-,. Degradation caused by neutrophils from the synovial fluid of rheumatoid arthritis patients was also inhibited by wortmannin. These data demonstrate that the proteoglycan degradative activity of neutrophils required PI3-K but not PKC or the ERK1/ERK2/ERK5 cascades and was insensitive to increases in intracellular cAMP concentrations. [source] Kinetics of inhibition of peroxidase activity of myeloperoxidase by quercetinINTERNATIONAL JOURNAL OF CHEMICAL KINETICS, Issue 7 2008Tatjana Momi The inhibition of myeloperoxidase (MPO), isolated from human neutrophils, by quercetin was investigated by following peroxidase activity of the enzyme using o -dianisidine as the substrate. The inhibition parameters (IC50) were obtained by graphical analysis of the inhibition curves. A reaction mechanism, which involved the enzyme inhibition by quercetin and H2O2 in excess, was proposed. The rate and equilibrium constants for the proposed reaction path were calculated from experimental data. Kinetic analysis in noninhibiting H2O2 concentration range in the absence and the presence of quercetin revealed that the reaction mechanism underwent Michaelis,Menten kinetics. K and V values indicated that quercetin was a mixed inhibitor of MPO activity. The initial reaction rates were recalculated using the obtained results. Calculated curves fitted the experimental results within the range of experimental error. © 2008 Wiley Periodicals, Inc. Int J Chem Kinet 40: 384,394, 2008 [source] Microinjected neutrophils retain the ability to take up bacteriaJOURNAL OF ANATOMY, Issue 5 2002M. M. Bird It is now possible to microinject protein to probe specific biochemical pathways and/or cell functions in small cells such as human neutrophils (Bird et al. J.Anat.198, 2001). We have shown that these cells retain their ability to modify their F-actin cytoskeleton following the microinjection procedure. The principal task of neutrophils is to hunt and kill bacteria by responding to chemotactic gradients which cause them to extend actin rich pseudopodia in the direction of the highest concentration of these molecules. On reaching their target the neutrophils make tight contact with the bacteria and phagocytosis ensues. Here we address the question of whether or not the microinjected cells are still able to maintain their normal phagocytic activities. Human neutrophils maintained in culture for 20 mins were confronted with Staphylococcus aureus (1 × 104 cells/mL) for 5 min and then injected with rat IgG as an exogenous protein that also serves as a marker for injected cells. After 30 min the cells were fixed for fluorescence or confocal microscopy in 3.7% formaldehyde and permeabilised for 5 min (0.2% Triton X-100 in PBS). They were then incubated for 45 min in 2.5 µL FITC-anti rat IgG and 1 µL TRITC-phalloidin (to show the F-actin cytoskeleton), in 996.5 µL of PBS, washed 6 times in PBS and mounted on slides in 5 µL Mowiol containing a grain of antiquench. For TEM cells were fixed in 1.5% glutaraldehyde in cacodylate buffer for 3 min at room temperature and then washed in 0.2 m cacodylate buffer 6 times before incubation with 1 mm NiCl2 and SIGMA fast DAB peroxidase tablets for 30 min. The cells were postfixed in a 2% solution of osmium tetroxide for 30 min, dehydrated through a series of graded ethanols, and embedded and sectioned for TEM. By TEM the injected neutrophils were observed to have taken up bacteria into vacuoles of varying size. At the earliest stages of this process, prior to and immediately following the initial release of granular contents and the initiation of mechanisms to rapidly destroy bacteria, the bacteria fitted more tightly in the vacuoles than at later stages. Injected neutrophils commonly contained several bacteria; more than one bacterium was frequently located within a single vacuole of substantial size. Confocal laser microscopic observations confirmed that cells containing ingested bacteria also contained IgG. Thus injected cells not only survive the microinjection procedure but also retain their ability to take up bacteria and initiate the digestive process. [source] Free radical,scavenging activity and DNA damaging potential of auxins IAA and 2-methyl-IAA evaluated in human neutrophils by the alkaline comet assayJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 3 2010Branka Salopek-Sondi Auxins, of which indole-3-acetic acid (IAA) is the most widespread representative, are plant hormones. In addition to plants, IAA also naturally occurs in humans in micromolar concentrations. In the presence of peroxidase, indolic auxins are converted to cytotoxic oxidation products and have thus been proposed for use in gene-directed enzyme/prodrug tumor therapy. Since data on the genotoxicity of IAA and its derivatives are not consistent, here we investigate the early DNA damaging effects (2-h treatment) of the auxins, IAA, and 2-methyl-indole-3-acetic acid (2-Me-IAA) by the alkaline comet assay and compare them with their free radical,scavenging activity measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Human neutrophils are chosen as the test system since they possess inherent peroxidase activity. The results of the comet assay indicate an increase in DNA damage in a dose-dependent manner up to 1.00 mM of both auxins. Generally, IAA applied in the same concentration had greater potential to damage DNA in human neutrophils than did 2-Me-IAA. The genotoxicities of the two examined auxins are negatively correlated with their antioxidant activities, as measured by the DPPH assay; 2-Me-IAA showed a higher antioxidant capacity than did IAA. We assume that differences in the molecular structure of the tested auxins contributed to differences in their metabolism, in particular, with respect to interactions with peroxidases and other oxidative enzymes in neutrophils. However, the exact mechanisms have to be elucidated in future studies. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:165,173, 2010; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20323 [source] Signal transduction and functional changes in neutrophils with agingAGING CELL, Issue 4 2004Tamas Fulop Summary It is well known that the immune response decreases during aging, leading to a higher susceptibility to infections, cancers and autoimmune disorders. Most widely studied have been alterations in the adaptive immune response. Recently, the role of the innate immune response as a first-line defence against bacterial invasion and as a modulator of the adaptive immune response has become more widely recognized. One of the most important cell components of the innate response is neutrophils and it is therefore important to elucidate their function during aging. With aging there is an alteration of the receptor-driven functions of human neutrophils, such as superoxide anion production, chemotaxis and apoptosis. One of the alterations underlying these functional changes is a decrease in signalling elicited by specific receptors. Alterations were also found in the neutrophil membrane lipid rafts. These alterations in neutrophil functions and signal transduction that occur during aging might contribute to the significant increase in infections in old age. [source] Applications of gold cluster compounds in immunocytochemistry and correlative microscopy: comparison with colloidal goldJOURNAL OF MICROSCOPY, Issue 3 2000J. M. Robinson In this review, we discuss the immunocytochemical literature with respect to a comparison between conventional colloidal gold and gold cluster compounds as immunoprobes. The relative advantages and disadvantages of each of these types of particle for immunocytochemical applications are discussed. We present results from our own laboratories and those of others on the comparison of these immunoprobes in selected experimental situations. These results show the use of gold cluster compounds at both light and electron microscope levels. At the ultrastructural level, gold cluster compounds have been used in pre-embedding labelling of cultured cells, and for labelling of ultrathin cryosections and freeze-fracture preparations. Recently, fluorescently tagged gold cluster compounds have become available. Using ultrathin cryosections of human neutrophils as a model system, we demonstrate that a single immunoprobe (i.e. a fluorescently tagged gold cluster compound) is a robust probe for correlative fluorescence and electron microscopy. [source] Hybrid ,/,3 -peptides with proteinogenic side chains. monosubstituted analogues of the chemotactic tripeptide For-Met-Leu-Phe-OMeJOURNAL OF PEPTIDE SCIENCE, Issue 8 2004Cesare Giordano Abstract The ,/,3 -mixed tripeptides R-CO-,3 -HMet-Leu-Phe-OMe (1a,b), R-CO-Met-,3 -HLeu-Phe-OMe (2a,b) and R-CO-Met-Leu-,3 -HPhe-OMe (3a,b) (a, R = tert -butyloxy-; b, R = H,), analogues of the potent chemoattractant For-Met-Leu-Phe-OMe, have been synthesized by classical solution methods and fully characterized. The activities of the new analogues as chemoattractants, superoxide anion producers and lysozyme releasers have been determined on human neutrophils. Whereas all of the three N -formyl derivatives are significantly less active than the parent tripeptide as chemoattractants, compound 1b has been found to be highly active as a superoxide anion producer and 3b as a lysozyme releaser. The results show that the replacement of the native Leu residue at the central position is, in each of the examined cases, the least favourable modification. The three N -Boc derivatives are, as expected, devoid of activity as agonists, but they are all good inhibitors of chemotaxis. Information on the solution conformation has been obtained by examining the involvement of the NH groups in intramolecular H-bonds using 1H NMR. The conformation of the N -Boc analogue 1a has also been determined in the crystal state by x-ray diffraction analysis. The molecule is extended at the ,3 -HMet residue (,1 = ,87°;,1 = 172°;,1 = 126° ) and no intramolecular H-bond is present. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd. [source] Probing structural requirements of fMLP receptor: On the size of the hydrophobic pocket corresponding to residue 2 of the tripeptideJOURNAL OF PEPTIDE SCIENCE, Issue 2 2002Susanna Spisani Abstract The conformationally constrained f- L -Met-Acnc- L -Phe-OMe (n = 4,9,12) tripeptides, analogues of the chemoattractant f- L -Met- L -Leu- L -Phe-OH, were synthesized in solution by classical methods and fully characterized. These compounds and the published f- L -Met-Xxx- L -Phe-OMe (Xxx = Aib and Acnc where n = 3, 5,8) analogues were compared to determine the combined effect of backbone preferred conformation and side-chain bulkiness at position 2 on the relation of 3D-structure to biological activity. A conformational study of all the analogues was performed in solution by FT-IR absorption and 1H-NMR techniques. In parallel, each peptide was tested for its ability to induce chemotaxis, superoxide anion production and lysozyme secretion from human neutrophils. The biological and conformational data are discussed in relation to the proposed model of the chemotactic receptor on neutrophils, in particular of the hydrophobic pocket accommodating residue 2 of the tripeptide. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd. [source] Calprotectin release from human neutrophils is induced by Porphyromonas gingivalis lipopolysaccharide via the CD-14,Toll-like receptor,nuclear factor ,B pathwayJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2003Jun-ichi Kido Objectives:, Calprotectin is a cytosolic protein with antibacterial action in leukocytes and its level increases in some inflammatory diseases, including periodontal diseases, rheumatoid arthritis and ulcerative colitis. Recently, we found that the lipopolysaccharide of Porphyromonas gingivalis (P-LPS) induced calprotectin release from human neutrophils. P-LPS, a major virulence factor of periodontal pathogens, is known to induce the production and release of inflammatory cytokines through CD14, Toll-like receptor (TLR) and nuclear factor ,B (NF-,B). In the present study, we investigated whether calprotectin release by P-LPS is induced via the CD14,TLR,NF-,B pathway and the cellular mechanism of calprotectin release in human neutrophils. Material and methods:, Human neutrophils were isolated from the peripheral blood of healthy donors and pre-incubated in medium containing antibodies against CD14, TLR2 and TLR4, or several inhibitors of NF-,B, microtubules and microfilaments, and then incubated with P-LPS. The calprotectin amount in the culture medium was determined using ELISA, and the nuclear extracts from cells were used for the examination of NF-,B binding activity using electrophoretic mobility shift assays. Results:, P-LPS increased calprotectin release from neutrophils and its induction was inhibited by anti-CD14 and anti-TLR2 antibodies, but not by two anti-TLR4 antibodies. NF-,B inhibitors suppressed P-LPS-induced NF-,B binding activity and calprotectin release. The inhibitors of microtubule and microfilament polymerization significantly decreased P-LPS-induced calprotectin release. Conclusion:, These results suggest that calprotectin release is induced by P-LPS via the CD14,TLR2,NF-,B signal pathway in human neutrophils and may be dependent on microtubule and microfilament systems. [source] | ||||||||||||||||||||||||||||||||||