Signal Transduction In Neutrophils Research Articles

Prognostic implications and functional enrichment analysis of LTB4R in patients with acute myeloid leukemia

To explore the expression patterns, prognostic implications, and biological role of leukotriene B4 receptor (LTB4R) in patients with acute myeloid leukemia (AML). We collected the data of mRNA expression levels and clinical information of patients with AML from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database for mRNA expression analyses, survival analyses, Cox regression analyses and correlation analyses using R studio to assess the expression patterns and prognostic value of LTB4R. The correlation of LTB4R expression levels with clinical characteristics of the patients were analyzed using UALCAN. The co-expressed genes LTB4R were screened from Linkedomics and subjected to functional enrichment analysis. A protein-protein interaction network was constructed using STRING. GSEA analyses of the differentially expressed genes (DEGs) were performed based on datasets from TCGA-LAML stratified by LTB4R expression level. We also collected peripheral blood mononuclear cells (PBMCs) from AML patients and healthy donors for examination of the mRNA expression levels of LTB4R and immune checkpoint genes using qRT-PCR. We also examined serum LTB4R protein levels in the patients using ELISA. The mRNA expression level of LTB4R was significantly increased in AML patients (4.898±1.220 vs 2.252±0.215, P < 0.001), and an elevated LTB4R expression level was correlated with a poor overall survival (OS) of the patients (P=0.004, HR=1.74). LTB4R was identified as an independent prognostic factor for OS (P=0.019, HR=1.66) and was associated with FAB subtypes, cytogenetic risk, karyotype abnormalities and NPM1 mutations. The co- expressed genes of LTB4R were enriched in the functional pathways closely associated with AML leukemogenesis, including neutrophil inflammation, lymphocyte activation, signal transduction, and metabolism. The DEGs were enriched in differentiation, activation of immune cells, and cytokine signaling. Examination of the clinical serum samples also demonstrated significantly increased expressions of LTB4R mRNA (P=0.044) and protein (P=0.008) in AML patients, and LTB4R mRNA expression was positively correlated with the expression of the immune checkpoint HAVCR2 (r= 0.466, P=0.040). LTB4R can serve as a novel biomarker and independent prognostic indicator of AML and its expression patterns provide insights into the crosstalk of leukemogenesis signaling pathways involving tumor immunity and metabolism.

A LncRNA-miRNA-mRNA ceRNA regulatory network based tuberculosis prediction model

The high incidence of tuberculosis (TB) has brought serious social burdens and it is urgent to explore the mechanism of TB development. This study was conducted to analyze the role of lncRNA-miRNA-mRNA regulatory network and its contained nodes involved in TB to identify crucial biomarkers for early diagnosis of TB. Long-noncoding RNAs (lncRNAs), messenger RNA (mRNAs) and microRNAs (miRNAs) expression profiles of TB patients and healthy individuals were downloaded from the GSE34608 dataset. Weighted gene co-expression network analysis (WGCNA) was performed to identified the key modules related to TB and the highly related mRNA-lncRNA pair in the module. Based on highly related mRNAs and lncRNAs in greenyellow module, lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network was constructed. The DE-mRNAs in the network were functionally enriched with Gene ontology (GO) and Gene set enrichment analysis (GSEA). Least absolute shrinkage and selection operator (LASSO) algorithm and receiver operating characteristic curve (ROC) were used to construct and evaluate the prediction model of TB. We identified 3267 DE-mRNAs, 484 DE-lncRNAs and 69 DE-miRNAs between the TB and healthy subjects, from which 8 DE-mRNAs, 14 DE-lncRNAs and 3 DE-miRNAs were used to construct the ceRNA network. The genes contained in the ceRNA network were mainly enriched in neutrophil mediated immune response, including neutrophil activation, degradation and signal transduction. ROC analysis revealed that has-miR-140–5p, has-miR-142–3p and the LASSO cox prediction model based on HMGA1 and CAPN1 have potential value for forecasting TB (AUC > 0.7). Hence, our study provides a new perspective from the lncRNA-miRNA-mRNA ceRNA regulatory network for TB diagnosis and treatment.

Effect of a Mushroom (Coriolus versicolor) Based Probiotic on the Expression of Toll-like Receptors and Signal Transduction in Goat Neutrophils

&lt;p class="1Body"&gt;Neutrophils recognize and destroy pathogens through activation of the Toll like receptor (TLR) system as part of the inflammatory response of innate immunity. The expression and modulation of genes in the TLR signaling pathway in caprine blood neutrophils was investigated. Following initial screening for infection, goats (N=15) were assigned to three groups of five (n=5) individuals. Goats were drenched daily with 10 mL of powdered CorPet (Mycology labs Inc) soaked in hot (treatment I) or cold (treatment II) sterile filtered endotoxin free water, for a 4-week period. A control group of five age-matched goats received sterile water (treatment III). Blood was collected weekly and analyzed for packed cell volume and white blood cell differential counts. At weeks 1 and 4 neutrophils were isolated, using differential centrifugation and hypotonic lysis of red blood cells. The concentration and purity of total RNA isolated using Trizol was determined on a Nanodrop spectrophotometer. The RETROscript kit was used to synthesize cDNA. The expression of 84 genes in the human TLR signaling pathway RT&lt;sup&gt;2&lt;/sup&gt; PCR Array was evaluated using real time PCR and the Livak method. The house keeping gene GAPDH was used to normalize the data. At week 1 untreated goats expressed 48 genes in the pathway. Goat neutrophils expressed 10 TLRs. Mushroom extracts modulated expression of and signaling by TLR. These results will help in the definition of the role of TLR expression in neutrophils and its contribution to goat innate immunity. Further this may aid in the design of therapeutics for goat health.&lt;/p&gt;

Mass spectrometric phosphoproteome analysis of small-sized samples of human neutrophils

BackgroundGlobal analysis of stimulus-dependent changes in the neutrophil phosphoproteome will improve the understanding of neutrophil signal transduction and function in diverse disease settings. However, gel-free phosphoproteomics of neutrophils in clinical studies is hampered by limited sample amounts and requires protein extract stability, efficient tryptic digestion and sensitive phosphopeptide enrichment in a protease-rich environment. For development of an appropriate workflow, we assessed neutrophil protein stability in urea-based lysis buffers and determined feasibility of gel-free phosphoproteomic analyses using polymer-based metal ion affinity capture (PolyMAC). MethodsWestern blotting, phosphopeptide enrichment and mass spectrometric analyses of samples of neutrophils were performed. ResultsDegradation of proteins in neutrophil extracts was observed after preparation with a urea-containing lysis buffer and could be prevented by addition of highly concentrated protease inhibitors. Subsequent tryptic digestion and PolyMAC-based phosphopeptide enrichment proved efficient with accordingly prepared neutrophil samples. Applying the new workflow, formyl–methionyl–leucyl–phenylalanine-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was detected after gel-free and gel-based phosphoproteomic analyses as proof of principle from 20ml of whole blood. Furthermore, phosphorylation of other ERK1/2 pathway-associated proteins was monitored. ConclusionWe provide a workflow for efficient, gel-free phosphoproteome analyses with small-sized neutrophil samples, suitable for application in clinical studies.

Mechanotransduction in neutrophil activation and deactivation

Mechanotransduction refers to the processes through which cells sense mechanical stimuli by converting them to biochemical signals and, thus, eliciting specific cellular responses. Cells sense mechanical stimuli from their 3D environment, including the extracellular matrix, neighboring cells and other mechanical forces. Incidentally, the emerging concept of mechanical homeostasis,long term or chronic regulation of mechanical properties, seems to apply to neutrophils in a peculiar manner, owing to neutrophils' ability to dynamically switch between the activated/primed and deactivated/deprimed states. While neutrophil activation has been known for over a century, its deactivation is a relatively recent discovery. Even more intriguing is the reversibility of neutrophil activation and deactivation. We review and critically evaluate recent findings that suggest physiological roles for neutrophil activation and deactivation and discuss possible mechanisms by which mechanical stimuli can drive the oscillation of neutrophils between the activated and resting states. We highlight several molecules that have been identified in neutrophil mechanotransduction, including cell adhesion and transmembrane receptors, cytoskeletal and ion channel molecules. The physiological and pathophysiological implications of such mechanically induced signal transduction in neutrophils are highlighted as a basis for future work. This article is part of a Special Issue entitled: Mechanobiology.

Intracellular signalling during neutrophil recruitment.

Recruitment of leucocytes such as neutrophils to the extravascular space is a critical step of the inflammation process and plays a major role in the development of various diseases including several cardiovascular diseases. Neutrophils themselves play a very active role in that process by sensing their environment and responding to the extracellular cues by adhesion and de-adhesion, cellular shape changes, chemotactic migration, and other effector functions of cell activation. Those responses are co-ordinated by a number of cell surface receptors and their complex intracellular signal transduction pathways. Here, we review neutrophil signal transduction processes critical for recruitment to the site of inflammation. The two key requirements for neutrophil recruitment are the establishment of appropriate chemoattractant gradients and the intrinsic ability of the cells to migrate along those gradients. We will first discuss signalling steps required for sensing extracellular chemoattractants such as chemokines and lipid mediators and the processes (e.g. PI3-kinase pathways) leading to the translation of extracellular chemoattractant gradients to polarized cellular responses. We will then discuss signal transduction by leucocyte adhesion receptors (e.g. tyrosine kinase pathways) which are critical for adhesion to, and migration through the vessel wall. Finally, additional neutrophil signalling pathways with an indirect effect on the neutrophil recruitment process, e.g. through modulation of the inflammatory environment, will be discussed. Mechanistic understanding of these pathways provide better understanding of the inflammation process and may point to novel therapeutic strategies for controlling excessive inflammation during infection or tissue damage.

Lidocaine Reduces Neutrophil Recruitment by Abolishing Chemokine-Induced Arrest and Transendothelial Migration in Septic Patients

The inappropriate activation, positioning, and recruitment of leukocytes are implicated in the pathogenesis of multiple organ failure in sepsis. Although the local anesthetic lidocaine modulates inflammatory processes, the effects of lidocaine in sepsis are still unknown. This double-blinded, prospective, randomized clinical trial was conducted to investigate the effect of lidocaine on leukocyte recruitment in septic patients. Fourteen septic patients were randomized to receive either a placebo (n = 7) or a lidocaine (n = 7) bolus (1.5 mg/kg), followed by continuous infusion (100 mg/h for patients >70 kg or 70 mg/h for patients <70 kg) over a period of 48 h. Selectin-mediated slow rolling, chemokine-induced arrest, and transmigration were investigated by using flow chamber and transmigration assays. Lidocaine treatment abrogated chemokine-induced neutrophil arrest and significantly impaired neutrophil transmigration through endothelial cells by inhibition of the protein kinase C-θ while not affecting the selectin-mediated slow leukocyte rolling. The observed results were not attributable to changes in surface expression of adhesion molecules or selectin-mediated capturing capacity, indicating a direct effect of lidocaine on signal transduction in neutrophils. These data suggest that lidocaine selectively inhibits chemokine-induced arrest and transmigration of neutrophils by inhibition of protein kinase C-θ while not affecting selectin-mediated slow rolling. These findings may implicate a possible therapeutic role for lidocaine in decreasing the inappropriate activation, positioning, and recruitment of leukocytes during sepsis.

Two new synthetic peptides from the N‐domain of CEACAM1 (CD66a) stimulate neutrophil adhesion to endothelial cells

Four members of the carcinoembryonic antigen family, CEACAMs 1, 3, 6, and 8, are expressed on human neutrophils and can trigger an activation signal that increases neutrophil adhesion to human umbilical vein endothelial cell (HUVEC) monolayers. To identify active sites on CEACAM1, we previously performed molecular modeling using IgG and CD4 as models, and 28 peptides of 14 amino acids in length were synthesized that were predicted to be present at loops and turns between β-sheets. Three peptides, each from the N-terminal domain, increased neutrophil adhesion to HUVEC monolayers and upregulated cell-surface CD11b/CD18 expression on neutrophils. In our earlier study, one N-domain peptide (CD66a-7) was not successfully synthesized, and another N-domain peptide (CD66a-6) was not soluble in the assay system. In the present study, we have now successfully synthesized CD66a-7, and a new peptide (CD66a-6L), that is a modification of the peptide that was insoluble in the earlier study. Both of these new peptides increased neutrophil adhesion to HUVEC monolayers. Importantly, the amino acid sequence of CD66a-7 is identical to the homologous peptides from CEACAMs 3, 5, and 6, but differs from the homologous peptide of CEACAM8, which was not active in this system. CD66a-6L is identical to the homologous peptide from CEACAM6. The data suggest that peptide motifs from at least five regions of the N-terminal domain of CEACAM1 are involved in the interaction of CEACAM1 with other ligands and can initiate signal transduction in neutrophils. Some of these active peptides are identical to homologous regions of other CEACAMs.

The effect of essential oil extracted from Nigella sativa (L.) seeds on human neutrophil functions

This study was aimed to investigate the effect of essential oil on human neutrophil (HN) functions. The neutrophils were isolated on percoll gradients, counted, and tested for viability using the trypan blue exclusion method. The chemotactic response was based on a multiple blind well assay system. The control movement and chemotactic response of neutrophils to 0.1 µM fMLP were reduced at a dose-dependent manner. The essential oil significantly inhibited neutrophil chemotaxis from 0.05 to 0.5 mg mL−1. The inhibitory concentrations (IC50) showing 50% inhibition to induced neutrophil chemotaxis, and control movement were 0.08 and 0.07 mg mL−1, respectively. The human neutrophil elastase secretion was inhibited by essential oil at a concentration dependent manner from 0.5 to 2.5 mg mL−1. The components of essential oil are potent inhibitors for polymorpho nuclear leukocytes functions. The observed inhibition of neutrophil functions occurred via intracellular pathway. Active serine protease could be essential for neutrophil responding process and/or signal transduction pathways.

Adhesive Dynamics Simulation of Neutrophil Arrest with Stochastic Activation

The transition from rolling to firm adhesion is a key step in the adhesion cascade that permits a neutrophil to exit the bloodstream and make its way to a site of inflammation. In this work, we construct an integrated model of neutrophil activation and arrest that combines a biomechanical model of neutrophil adhesion and adhesive dynamics, with fully stochastic signal transduction modeling, in the form of kinetic Monte Carlo simulation within the microvilli. We employ molecular binding parameters gleaned from the literature and from simulation of cell-free rolling mediated by selectin molecules. We create a simplified model of lymphocyte function-associated antigen-1 activation that links P-selectin glycoprotein ligand-1 ligation to integrin activation. The model utilizes an energy profile of various integrin activation states drawn from literature data and permits manipulation of signal diffusivity within the microvillus. Our integrated model recreates neutrophil arrest within physiological timescales, and we demonstrate that increasing signal diffusivity within a microvillus accelerates arrest. If the energy barrier between free unactivated and free activated lymphocyte function-associated antigen-1 increases, the period of rolling before arrest increases. We further demonstrate that, within our model, modification of endothelial ligand surface densities can control arrest. In addition, the relative concentrations of signaling molecules control the fractional activation of the overall signaling pathway and the rolling time to arrest. This work presents the first, to our knowledge, fully stochastic model of neutrophil activation, which, though simplified, can recapitulate significant physiological details of neutrophil arrest yet retains the capacity to incorporate additional information regarding mechanisms of neutrophil signal transduction as they are elucidated.

The effect of anti-neutrophil cytoplasm autoantibodies on the signal transduction in human neutrophils

The effect of anti-neutrophil cytoplasm autoantibodies (ANCA) on neutrophil activation was studied by pre-incubating neutrophils with IgG and F(ab')2 prepared from ANCA patients with Wegener's granulomatosis or microscopic polyarteritis. We measured the generation of inositol triphosphate (IP3) (a product of hydrolysis of membrane phospholipid which acts as a second intracellular messenger), and the translocation of protein kinase C (PKC) upon stimulation by a chemotactic peptide, fMet-Leu-Phe (fMLP). ANCA+ F(ab')2 did not induce a significant increase in IP3 generation. Nonetheless, ANCA+ F(ab')2 and ANCA+ IgG pretreatment of human neutrophils reduced the production of inositol phosphates upon subsequent fMLP stimulation compared with experiments performed when cells were pretreated with F(ab')2 and IgG prepared from ANCA- healthy subjects. A significantly reduced generation of IP3 and inositol biphosphate (IP2) was observed. ANCA+ F(ab')2 pretreatment of neutrophils inhibited fMLP-stimulated IP3 generation in a dose-dependent manner. The membrane-bound PKC activity upon stimulation by FMLP and PMA was reduced in neutrophils pretreated with ANCA+ F(ab')2 and IgG. These results indicate that ANCA affect in vitro signal transduction (IP3 generation, and translocation of PKC) in human neutrophils. Apparently, further activation of signal transduction by chemotactic peptide is significantly blunted in cells pre-incubated with ANCA+ F(ab')2 but not with F(ab')2 from healthy controls.

Eicosapentaenoic acid modulates neutrophil leukotriene B4 receptor expression in cystic fibrosis.

In patients with cystic fibrosis (CF), high intrapulmonary concentrations of the neutrophil chemotaxin leukotriene B4 (LTB4) are associated with specific reduction of LTB4-induced chemotaxis of circulating neutrophils. The chemotactic abnormality is partially corrected by dietary supplementation with eicosapentaenoic acid (EPA). LTB4-induced neutrophil chemotaxis is mediated by specific, high-affinity, cell surface LTB4 receptors. The hypotheses that neutrophil LTB4 receptors are down-regulated in CF, and that EPA normalizes receptor expression, were tested by measuring the number (Rmax) and affinity (Kd) of LTB4 receptors on neutrophils from eight CF patients before and after EPA (6 weeks of 2.7 g/day), and from nine normal individuals. High-affinity receptor Rmax was depressed in CF patients (0.6 +/- 0.2 x 10(4)/cell (mean +/- s.d.) versus 1.8 +/- 0.7 x 10(4)/cell in normals), but corrected to normal (2.0 +/- 1.9 x 10(4)/cell) after EPA. High-affinity receptor Kd was depressed in CF patients (0.4 +/- 0.3 nM versus 1.4 +/- 0.5 nM in normals), and also corrected to normal with EPA (1.4 +/- 1.2 nM). Low-affinity receptors were depressed, but did not change significantly with EPA. These results indicate that neutrophil responses in chronic inflammatory lung disease can be influenced directly by LTB4 receptor modulation, and that this effect of EPA predominates over alterations in neutrophil signal transduction in situations of chronic exposure to LTB4.

Role of Toll-like receptor 4 in hyperoxia-induced lung inflammation in mice

Prolonged exposure to hyperoxia causes lung inflammation, but the role of Toll-like receptor 4 (TLR4) in hyperoxia-induced signal transduction remains unclear. We evaluated neutrophil accumulation, signal transduction and cytokine production during hyperoxia, comparing TLR4 mutant (C3H/HeJ) and wild type (C3H/HeN) mice. The mice were exposed to 80% oxygen in a hyperoxic chamber for 0 (control), 48, or 96 h. After the exposure, bronchoalveolar lavage (BAL) was performed for differential cell counting and cytokine measurement. In lung homogenate, activation of NF-kappaB and STAT1 was also examined. In C3H/HeJ mice, hyperoxia-induced neutrophil accumulation in BAL fluid was significantly decreased compared with C3H/HeN. Hyperoxia for 96 h caused NF-kappaB translocation in C3H/HeN mice, which was significantly attenuated in C3H/HeJ mice (p < 0.05). In contrast, STAT1 activation occurred as early as after 48 h of oxygen exposure, which did not differ between the two strains. The levels of TNF-alpha, IL-6, and KC in BAL fluid were increased after oxygen exposure, which was suppressed by the lack of TLR4 signaling. These results suggest that TLR4-dependent NF-kB activation may be an important process of the upregulation of proinflammatory mediators and subsequent neutrophil accumulation into the lung during hyperoxia.

Costimulatory Effects of Complement Receptor Type 3 and Fc Receptor for IgG (Fc.GAMMA.R) on Superoxide Production and Signal Transduction in Bovine Neutrophils

The present study evaluated the costimulatory effects of complement receptor type 3 (CR3) and Fc receptor for IgG (FcgammaR) on superoxide production and intracellular signal transduction in bovine neutrophils. Stimulation with opsonized zymosan (OPZ) and heat-aggregated bovine IgG (Agg-IgG) resulted in much greater superoxide production and chemiluminescent (CL) responses in normal neutrophils compared with those stimulated with OPZ or Agg-IgG only. Superoxide production and CL response were closely associated with the stimulant-induced rise of the intracellular calcium ([Ca2+]i) concentration, amount of tyrosine phosphorylated 100 kDa protein, and activation of p38 mitogen-activated protein kinase (p38 MAPK). No costimulatory effect was found for these receptors on superoxide production in CR3-deficient neutrophils. Costimulation of CR3 and FcgammaR on bovine neutrophils leads to enhancement of superoxide production and their signaling pathways and appears to be associated with enhancement of neutrophil functions.

Isolation and characterization of the potential receptor for wheat germ agglutinin from human neutrophils

Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.

Chemical inhibitors of TNF signal transduction in human neutrophils point to distinct steps in cell activation

Chemical screening identified three small compounds that selectively inhibited activation of the respiratory burst (RB) of human neutrophils in response to tumor necrosis factor (TNF) and formylated peptide but not phorbol ester and spared the ability of neutrophils to kill bacteria. These compounds partially inhibited TNF-triggered cytoskeletal rearrangements without blocking adhesion or transmigation of polymorphonuclear neutrophils through TNF-activated monolayers of endothelial cells. The compounds were nontoxic to neutrophils and endothelial cells. They had no direct inhibitory effect on the tyrosine kinases Src, Syk, or Pyk2. However, their differential effects on cell spreading, bacteria-induced RB, TNF-induced degranulation, TNF-induced protein tyrosine phosphorylation, and TNF-induced Syk activation suggested that each may act on different elements of neutrophil signaling pathways.

Molecular Evolution of CXCR1, a G Protein-Coupled Receptor Involved in Signal Transduction of Neutrophils

Human neutrophils are a type of white blood cell, which forms an early line of defense against bacterial infections. Neutrophils are highly responsive to the chemokine, interleukin-8 (IL-8) due to the abundant distribution of CXCR1, one of the IL-8 receptors on the neutrophil cell surface. As a member of the GPCR family, CXCR1 plays a crucial role in the IL-8 signal transduction pathway in neutrophils. We sequenced the complete coding region of the CXCR1 gene in worldwide human populations and five representative nonhuman primate species. Our results indicate accelerated protein evolution in the human lineage, which was likely caused by Darwinian positive selection. The sliding window analysis and the codon-based neutrality test identified signatures of positive selection at the N-terminal ligand/receptor recognition domain of human CXCR1.

Calcium-sensing soluble adenylyl cyclase mediates TNF signal transduction in human neutrophils

Through chemical screening, we identified a pyrazolone that reversibly blocked the activation of phagocyte oxidase (phox) in human neutrophils in response to tumor necrosis factor (TNF) or formylated peptide. The pyrazolone spared activation of phox by phorbol ester or bacteria, bacterial killing, TNF-induced granule exocytosis and phox assembly, and endothelial transmigration. We traced the pyrazolone's mechanism of action to inhibition of TNF-induced intracellular Ca2+ elevations, and identified a nontransmembrane (“soluble”) adenylyl cyclase (sAC) in neutrophils as a Ca2+-sensing source of cAMP. A sAC inhibitor mimicked the pyrazolone's effect on phox. Both compounds blocked TNF-induced activation of Rap1A, a phox-associated guanosine triphosphatase that is regulated by cAMP. Thus, TNF turns on phox through a Ca2+-triggered, sAC-dependent process that may involve activation of Rap1A. This pathway may offer opportunities to suppress oxidative damage during inflammation without blocking antimicrobial function.

Lipoxins and aspirin-triggered lipoxins in neutrophil adhesion and signal transduction

Lipoxin A 4 (LXA 4) and aspirin-triggered 15-epi-LXA 4 (ATL) are emerging as endogenous braking signals for neutrophil-mediated tissue injury. LXA 4 and ATL and their metabolically stable analogues display potent inhibitory actions in human isolated cells and blood, including attenuation of expression of adhesion molecules on leukocytes and endothelial cells, neutrophil adhesion to endothelial cells and platelets under shear, and IL-8 production, key events of the acute inflammatory response. The underlying molecular mechanisms include interference with MAPK signaling pathways, modulation of the oxidative chemistry of superoxide, NO and ONOO −, inhibition of activation of NF- κB and AP-1, and consequently the expression of interleukin-8 and likely other pro-inflammatory genes. Collectively, these results add to the profile of LXA 4/ATL rapid actions that contribute to “stop signaling” involved in regulating neutrophil functions during acute inflammation and suggest that aspirin inhibits neutrophil accumulation through triggering the synthesis of 15-epi-LXA 4.

Neutrophils Exhibit Rapid Agonist-induced Increases in Protein-associated O-GlcNAc

A variety of cytoplasmic and nuclear proteins can be modified on serine and threonine residues by O-linked beta-N-acetylglucosamine (O-GlcNAc), although the effects of this modification on protein and cellular functions are not completely defined. The sugar donor for the O-GlcNAc transferase that catalyzes this post-translational modification is UDP-N-acetylglucosamine (UDP-GlcNAc), a product of the hexosamine biosynthesis pathway (HBP). Here, the dynamics of the O-GlcNAc modification are examined in the physiological context of agonist-induced signal transduction using neutrophils. Formylated Met-Leu-Phe (fMLF) is shown to stimulate a rapid and transient increase in protein O-GlcNAcylation in both immunoblot and immunofluorescence imaging assays using O-GlcNAc-specific antibodies. In high performance liquid chromatography analyses of HBP metabolic activity, short term exposure to an exogenous substrate of the HBP, glucosamine (GlcNH(2)), leads to increased GlcNH(2) 6-phosphate and then UDP-GlcNAc levels. The GlcNH(2) treatments also increase O-GlcNAcylation and augment the aforementioned fMLF-associated increase. In functional assays, GlcNH(2) pre-treatment selectively augments fMLF-induced chemotaxis but has little effect on respiratory burst activity. Furthermore, augmenting levels of O-GlcNAc in the absence of agonist is sufficient to stimulate chemotaxis. These data demonstrate that neutrophils possess a functionally significant O-GlcNAcylation pathway that is robustly induced by stimulation with agonist. We propose that O-GlcNAcylation plays an important role in rapid and dynamic neutrophil signal transduction, especially with respect to chemotaxis.