Search

Introduction: G-protein coupled receptors (GPCRs) expressed on neutrophils regulate their mobilization from the bone marrow into the blood, their half-live in the circulation, and their pro- and anti-inflammatory activities during inflammation. Chronic kidney disease (CKD) is associated with systemic inflammatory responses, and neutrophilia is a hallmark of CKD onset and progression. Nonetheless, the role of neutrophils in CKD is currently unclear., Methods: Blood and renal tissue were collected from non-dialysis CKD (grade 3 - 5) patients to evaluate GPCR neutrophil expressions and functions in CKD development., Results: CKD patients presented a higher blood neutrophil-to-lymphocyte ratio (NLR), which was inversely correlated with the glomerular filtration rate (eGFR). A higher frequency of neutrophils expressing the senescent GPCR receptor (CXCR4) and activation markers (CD18 + CD11b + CD62L + ) was detected in CKD patients. Moreover, CKD neutrophils expressed higher amounts of GPCR formyl peptide receptors (FPR) 1 and 2, known as neutrophil pro- and anti-inflammatory receptors, respectively. Cytoskeletal organization, migration, and production of reactive oxygen species (ROS) by CKD neutrophils were impaired in response to the FPR1 agonist (fMLP), despite the higher expression of FPR1. In addition, CKD neutrophils presented enhanced intracellular, but reduced membrane expression of the protein Annexin A1 (AnxA1), and an impaired ability to secrete it into the extracellular compartment. Secreted and phosphorylated AnxA1 is a recognized ligand of FPR2, pivotal in anti-inflammatory and efferocytosis effects. CKD renal tissue presented a low number of neutrophils, which were AnxA1 + ., Conclusion: Together, these data highlight that CKD neutrophils overexpress GPCRs, which may contribute to an unbalanced aging process in the circulation, migration into inflamed tissues, and efferocytosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Scharf, Sandri, Rizzetto, Xavier, Grosso, Correia-Silva, Farsky, Gil and Farsky.)

Gradual loss of neuronal structure and function due to impaired blood-brain barrier (BBB) and neuroinflammation are important factors in multiple sclerosis (MS) progression. Our previous studies demonstrated that the C16 peptide and angiopoietin 1 (Ang-1) compound (C + A) could modulate inflammation and vascular protection in many models of MS. In this study, nanotechnology and a novel nanovector of the leukocyte chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) were used to examine the effects of C + A on MS. The acute experimental autoimmune encephalomyelitis (EAE) model of MS was established in Lewis rats. The C + A compounds were conjugated to control nano-carriers and fMLP-nano-carriers and administered to animals by intravenous injection. The neuropathological changes in the brain cortex and spinal cord were examined using multiple approaches. The stimulation of vascular injection sites was examined using rabbits. The results showed that all C + A compounds (C + A alone, nano-carrier C + A, and fMLP-nano-carrier C + A) reduced neuronal inflammation, axonal demyelination, gliosis, neuronal apoptosis, vascular leakage, and BBB impairment induced by EAE. In addition, the C + A compounds had minimal side effects on liver and kidney functions. Furthermore, the fMLP-nano-carrier C + A compound had better effects compared to C + A alone and the nano-carrier C + A. This study indicated that the fMLP-nano-carrier C + A could attenuate inflammation-related pathological changes in EAE and may be a potential therapeutic strategy for the treatment of MS and EAE.

The bacteria-derived formyl peptide fMet-Leu-Phe (fMLF) is a potent chemoattractant of phagocytes that induces chemotaxis at subnanomolar concentrations. At higher concentrations, fMLF inhibits chemotaxis while stimulating degranulation and superoxide production, allowing phagocytes to kill invading bacteria. How an agonist activates distinct cellular functions at different concentrations remains unclear. Using a bioluminescence resonance energy transfer-based FPR1 biosensor, we found that fMLF at subnanomolar and micromolar concentrations induced distinct conformational changes in FPR1, a Gi-coupled chemoattractant receptor that activates various phagocyte functions. Neutrophil-like HL-60 cells exposed to subnanomolar concentrations of fMLF polarized rapidly and migrated along a chemoattractant concentration gradient. These cells also developed an intracellular Ca 2+ concentration gradient. In comparison, high nanomolar and micromolar concentrations of fMLF triggered the PLC-β/diacyl glycerol/inositol trisphosphate pathway downstream of the heterotrimeric Gi proteins, leading to Ca 2+ mobilization from intracellular stores and Ca 2+ influx from extracellular milieu. A robust and uniform rise in cytoplasmic Ca 2+ level was required for degranulation and superoxide production but disrupted cytoplasmic Ca 2+ concentration gradient and inhibited chemotaxis. In addition, elevated ERK1/2 phosphorylation and β-arrestin2 membrane translocation were associated with diminished chemotaxis in the presence of fMLF above 1 nM. These findings suggest a mechanism for FPR1 agonist concentration-dependent signaling that leads to a switch from migration to bactericidal activities in phagocytes.

Three-dimensional tracking of cells is one of the most powerful methods to investigate multicellular phenomena, such as ontogenesis, tumor formation or wound healing. However, 3D tracking in a biological environment usually requires fluorescent labeling of the cells and elaborate equipment, such as automated light sheet or confocal microscopy. Here we present a simple method for 3D tracking large numbers of unlabeled cells in a collagen matrix. Using a small lensless imaging setup, consisting of an LED and a photo sensor only, we were able to simultaneously track ~3000 human neutrophil granulocytes in a collagen droplet within an unusually large field of view (>50 mm2) at a time resolution of 4 seconds and a spatial resolution of ~1.5 μm in xy- and ~30 μm in z-direction. The setup, which is small enough to fit into any conventional incubator, was used to investigate chemotaxis towards interleukin-8 (IL-8 or CXCL8) and N-formylmethionyl-leucyl-phenylalanine (fMLP). The influence of varying stiffness and pore size of the embedding collagen matrix could also be quantified. Furthermore, we demonstrate our setup to be capable of telling apart healthy neutrophils from those where a condition of inflammation was (I) induced by exposure to lipopolysaccharide (LPS) and (II) caused by a pre-existing asthma condition. Over the course of our experiments we have tracked more than 420.000 cells. The large cell numbers increase statistical relevance to not only quantify cellular behavior in research, but to make it suitable for future diagnostic applications, too., Competing Interests: The authors have declared that no competing interests exist.

Exaggerated neutrophil activation and formation of neutrophil extracellular traps (NETs) are reported in systemic sclerosis (SSc) but its involvement in SSc pathogenesis is not clear. In the present study we assessed markers of neutrophil activation and NET formation in SSc patients in relation to markers of inflammation and disease phenotype. Factors promoting neutrophil activation in SSc remain largely unknown. Among the neutrophil activating factors, mitochondrial-derived N-formyl methionine (fMet) has been reported in several autoinflammatory conditions. The aim of the current study is to assess whether SSc patients have elevated levels of fMet and the role of fMet in neutrophil-mediated inflammation on SSc pathogenesis. Markers of neutrophil activation (calprotectin, NETs) and levels of fMet were analyzed in plasma from two SSc cohorts (n=80 and n=20, respectively) using ELISA. Neutrophil activation assays were performed in presence or absence of formyl peptide receptor 1 (FPR1) inhibitor cyclosporin H. Elevated levels of calprotectin and NETs were observed in SSc patients as compared to healthy controls (p<0.0001) associating with SSc clinical disease characteristics. Further, SSc patients had elevated levels of circulating fMet as compared to healthy controls (p<0.0001). Consistent with a role for fMet-mediated neutrophil activation, fMet levels correlated with levels of calprotectin and NETs (r=0.34, p=0.002; r=0.29, p<0.01 respectively). Additionally, plasma samples from SSc patients with high levels of fMet induced de novo neutrophil activation through FPR1-dependent mechanisms. Our data for the first time implicates an important role for the mitochondrial component fMet in promoting neutrophil-mediated inflammation in SSc., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kuley, Stultz, Duvvuri, Wang, Fritzler, Hesselstrand, Nelson and Lood.)

A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.

Neutrophils sense and migrate through an enormous range of chemoattractant gradients through adaptation. Here, we reveal that in human neutrophils, calcium-promoted Ras inactivator (CAPRI) locally controls the GPCR-stimulated Ras adaptation. Human neutrophils lacking CAPRI ( capri kd ) exhibit chemoattractant-induced, nonadaptive Ras activation; significantly increased phosphorylation of AKT, GSK-3α/3β, and cofilin; and excessive actin polymerization. capri kd cells display defective chemotaxis in response to high-concentration gradients but exhibit improved chemotaxis in low- or subsensitive-concentration gradients of various chemoattractants, as a result of their enhanced sensitivity. Taken together, our data reveal that CAPRI controls GPCR activation-mediated Ras adaptation and lowers the sensitivity of human neutrophils so that they are able to chemotax through a higher-concentration range of chemoattractant gradients., Competing Interests: The authors declare no competing interest.

Two new isosarcophine derivatives, cherbonolides M ( 1 ) and N ( 2 ), were further isolated from a Formosan soft coral Sarcophyton cherbonnieri . The planar structure and relative configuration of both compounds were established by the detailed analysis of the IR, MS, and 1D and 2D NMR data. Further, the absolute configuration of both compounds was determined by the comparison of CD spectra with that of isosarcophine ( 3 ). Notably, cherbonolide N ( 2 ) possesses the unique cembranoidal scaffold of tetrahydrooxepane with the 12,17-ether linkage fusing with a γ -lactone. In addition, the assay for cytotoxicity of both new compounds revealed that they showed to be noncytotoxic toward the proliferation of A549, DLD-1, and HuCCT-1 cell lines. Moreover, the anti-inflammatory activities of both metabolites were carried out by measuring the N -formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced generation of superoxide anion and elastase release in the primary human neutrophils. Cherbonolide N ( 2 ) was found to reduce the generation of superoxide anion (20.6 ± 6.8%) and the elastase release (30.1 ± 3.3%) in the fMLF/CB-induced human neutrophils at a concentration of 30 μM.

Acute leukemias, the most common cancers in children, are characterized by excessive proliferation of malignant progenitor cells. As a consequence of impaired blood cell production, leukemia patients are susceptible to infectious complications-a major cause of non-relapse mortality. Neutrophil extracellular traps (NETs) are involved in various pathologies, from autoimmunity to cancer. Although aberrant NETs formation may be partially responsible for immune defects observed in acute leukemia, still little is known on the NET release in the course of leukemia. Here, we present the first comprehensive evaluation of NETs formation by neutrophils isolated from children with acute leukemia in different stages of the disease and treatment stimulated in vitro with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore (CI). NETs release was measured using quantitative fluorescent method and visualized microscopically. In this setting, NETs release was significantly impaired in leukemic children both at the diagnosis and during the treatment, and full restoration of neutrophil function was achieved only after successful completion of the leukemia treatment. We suggest that neutrophil function impairment may result from both disease- and treatment-related factors. In this context, deficient innate immune response observed in acute leukemia patients may be present regardless of neutrophil count and contribute to secondary immunodeficiency observed in this population.

Qin Pi ( Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8 E )-4''- O -methylligstroside ( 1 ), (8 E )-4''- O -methyldemethylligstroside ( 2 ), and 3'',4''-di- O -methyl-demethyloleuropein ( 3 ), have been isolated from the stem bark of Fraxinus chinensis , together with 23 known compounds ( 4 - 26 ). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8 E )-4''- O -methylligstroside ( 1 ), (8 E )-4''- O -methyldemethylligstroside ( 2 ), 3'',4''-di- O -methyldemethyloleuropein ( 3 ), oleuropein ( 6 ), aesculetin ( 9 ), isoscopoletin ( 11 ), aesculetin dimethyl ester ( 12 ), fraxetin ( 14 ), tyrosol ( 21 ), 4-hydroxyphenethyl acetate ( 22 ), and (+)-pinoresinol ( 24 ) exhibited inhibition (IC 50 ≤ 7.65 μg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1 , 9 , 11 , 14 , 21 , and 22 inhibited fMLP/CB-induced elastase release with IC 50 ≤ 3.23 μg/mL. In addition, compounds 2 , 9 , 11 , 14 , and 21 showed potent inhibition with IC 50 values ≤ 27.11 μM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines , tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1 , 9 , and 14 . Compounds 1 , 9 , and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1 , 9 , and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1 , 9 , and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.

Following nerve injury, disintegrated axonal mitochondria distal to the injury site release mitochondrial formylated peptides and DNA that can induce activation and inflammatory profiling of Schwann cells via formyl peptide receptor 2 (Fpr2) and toll-like receptor 9 (TLR9), respectively. We studied RT4 schwannoma cells to investigate the regulation of Fpr2 and TLR9 after stimulation with fMLF as a prototypical formylated peptide. RT4 cells were treated with fMLF at various concentrations and times with and without pretreatment with inhibitors (chloroquine for activated TLR9, PBP10 for Fpr2). Western blots of Fpr2, TLR9, p-p38, p-NFκB, and IL-6 were compared in relation to inflammatory profiling of RT4 cells and chemokine receptors (CCR2, CXCR4) as potential co-receptors of Fpr2. fMLF stimulation upregulated Fpr2 in RT4 cells at low concentrations (10 nM and 100 nM) but higher concentrations were required (10 µM and 50 µM) when the cells were pretreated with an activated TLR9 inhibitor. Moreover, the higher concentrations of fMLF could modulate TLR9 and inflammatory markers. Upregulation of Fpr2 triggered by 10 nM and 100 nM fMLF coincided with higher levels of chemokine receptors (CCR2, CXCR4) and PKCβ. Treating RT4 cells with fMLF, as an in vitro model of Schwann cells, uncovered Schwann cells' complex responses to molecular patterns of release from injured axonal mitochondria.

Aims: Takotsubo syndrome (TTS) episodes are primarily initiated by 'pulse' release of catecholamines inducing neutrophil infiltration and myocardial inflammation in susceptible individuals (largely ageing women). Evidence of myocardial inflammation and associated energetic impairment persists for ≥ 3 months post-acute TTS episodes, suggesting the existence of additional 'perpetuating' mechanisms. The effects of B-type natriuretic peptide (BNP) in suppressing superoxide (O 2 - ) release from neutrophils are transiently impaired in acute heart failure. We also evaluated the extent and duration of BNP-induced suppression of O 2 - release post-TTS., Methods and Results: TTS patients were studied acutely (n = 34) and 3 months thereafter (n = 13) and compared with control subjects (n = 25). O 2 - generation from neutrophils, triggered by N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate, and its suppression by BNP, were measured in vitro. Determinants of variability in BNP effect were sought via univariate and multivariate analyses. Relative to control subjects, in TTS patients, BNP suppression of both phorbol myristate acetate and N-formyl-methionyl-leucyl-phenylalanine-induced O 2 - release was impaired acutely (P < 0.05 for both); this did not improve over the 3-month recovery period, despite treatment with conventional anti-failure medication in 85% of patients. No significant correlates of BNP effect (other than TTS) were identified., Conclusions: (1) While TTS is associated with marked and prolonged release of BNP, there is virtually total loss of the ability of BNP to suppress neutrophil O 2 - release and its impact on tissue inflammation. (2) BNP responses do not recover for at least 3 months post-attacks, suggesting that this might contribute to perpetuation of myocardial inflammation in TTS patients., (© 2020 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

The importance of the intracellular Ca 2+ concentration ([Ca 2+ ] i ) in neutrophil function has been intensely studied. However, the role of the intracellular Na + concentration ([Na + ] i ) which is closely linked to the intracellular Ca 2+ regulation has been largely overlooked. The [Na + ] i is regulated by Na + transport proteins such as the Na + /Ca 2+ -exchanger (NCX1), Na + /K + -ATPase, and Na + -permeable, transient receptor potential melastatin 2 (TRPM2) channel. Stimulating with either N-formylmethionine-leucyl-phenylalanine (fMLF) or complement protein C5a causes distinct changes of the [Na + ] i . fMLF induces a sustained increase of [Na + ] i , surprisingly, reaching higher values in TRPM2 -/- neutrophils. This outcome is unexpected and remains unexplained. In both genotypes, C5a elicits only a transient rise of the [Na + ] i . The difference in [Na + ] i measured at t = 10 min after stimulation is inversely related to neutrophil chemotaxis. Neutrophil chemotaxis is more efficient in C5a than in an fMLF gradient. Moreover, lowering the extracellular Na + concentration from 140 to 72 mM improves chemotaxis of WT but not of TRPM2 -/- neutrophils. Increasing the [Na + ] i by inhibiting the Na + /K + -ATPase results in disrupted chemotaxis. This is most likely due to the impact of the altered Na + homeostasis and presumably NCX1 function whose expression was shown by means of qPCR and which critically relies on proper extra- to intracellular Na + concentration gradients. Increasing the [Na + ] i by a few mmol/l may suffice to switch its transport mode from forward (Ca 2+ -efflux) to reverse (Ca 2+ -influx) mode. The role of NCX1 in neutrophil chemotaxis is corroborated by its blocker, which also causes a complete inhibition of chemotaxis., (Copyright © 2020 Najder, Rugi, Lebel, Schröder, Oster, Schimmelpfennig, Sargin, Pethő, Bulk and Schwab.)

High-level reactive oxygen species (ROS) production in neutrophils is tightly regulated, as it can damage host cells. Neutrophils also undergo low-level ROS production when stimulated by cytokines or chemoattractants, but its biologic significance remains largely unknown. Voltage-gated proton channels (Hv1/VSOP) activity reportedly supports ROS production in neutrophils; however, we show here that Hv1/VSOP balances ROS production to suppress neutrophil directional migration in the presence of low concentrations of N-formyl-Met-Leu-Phe (fMLF). Neutrophils derived from Hvcn1 gene knockout mice produced more ROS than neutrophils from wild-type mice in the stimulation with fMLF at concentration of 1 µM and nonstimulus condition. They also exhibited stronger chemotactic responses to low concentrations of fMLF than did wild-type neutrophils. Receptor sensitivity to fMLF and evoked Ca 2+ responses did not differ between Hv1/VSOP-deficient and wild-type neutrophils. Activation of ERK, but not p38, was enhanced and prolonged during the increased ROS production seen after fMLF stimulation in Hv1/VSOP-deficient neutrophils. Inhibiting ROS production suppressed the enhanced ERK activation in Hv1/VSOP-deficient neutrophils and their directional migration. These results indicate that Hv1/VSOP balances ROS production to reduce ERK signaling and suppress excessive neutrophil migration in response to fMLF. Our findings thus reveal a novel role for ROS in the directional migration of neutrophils., (©2020 Society for Leukocyte Biology.)

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments., Competing Interests: The authors have declared that no competing interests exist.

Circulating neutrophils are, by necessity, quiescent and relatively unresponsive to acute stimuli. In regions of inflammation, mediators can prime neutrophils to react to acute stimuli with stronger proinflammatory, pathogen-killing responses. In neutrophils G protein-coupled receptor (GPCR)-driven proinflammatory responses, such as reactive oxygen species (ROS) formation and accumulation of the key intracellular messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), are highly dependent on PI3K-γ, a Ras-GTP, and Gβγ coincidence detector. In unprimed cells, the major GPCR-triggered activator of Ras is the Ras guanine nucleotide exchange factor (GEF), Ras guanine nucleotide releasing protein 4 (RasGRP4). Although priming is known to increase GPCR-PIP 3 signaling, the mechanisms underlying this augmentation remain unclear. We used genetically modified mice to address the role of the 2 RasGEFs, RasGRP4 and son of sevenless (SOS)1/2, in neutrophil priming. We found that following GM-CSF/TNFα priming, RasGRP4 had only a minor role in the enhanced responses. In contrast, SOS1/2 acquired a substantial role in ROS formation, PIP 3 accumulation, and ERK activation in primed cells. These results suggest that SOS1/2 signaling plays a key role in determining the responsiveness of neutrophils in regions of inflammation., (©2019 Society for Leukocyte Biology.)

Microtubules (MTs) are critically involved in the transport of material within cells, but their roles in chemotactic leukocyte motility and effector functions are still obscure. Resting neutrophils contain few MTs assembled in an MT organizing center (MTOC) behind their multilobular nuclei. Using a probe of real-time tubulin polymerization, SiR-tubulin, we found that neutrophils elongated their MTs within minutes in response to signals from the two prototypic chemotactic peptides, CXCL1 and fMLP. Taxol, a beta-tubulin binding and MT stabilizing drug, was found to abolish this CXCL1- and fMLP-stimulated MT polymerization. Nevertheless, taxol treatment as well as disruption of existing and de novo generated MTs did not impair neutrophil protrusion and squeezing through IL-1β-stimulated endothelial monolayers mediated by endothelial deposited CXCL1 and neutrophil CXCR2. Notably, CXCL1-dependent neutrophil TEM was not associated with neutrophil MT polymerization. Chemokinetic neutrophil motility on immobilized CXCL1 was also not associated with MT polymerization, and taxol treatment did not interfere with this motility. Nevertheless, and consistent with its ability to suppress MT polymerization induced by soluble CXCL1 and fMLP, taxol treatment inhibited neutrophil chemotaxis toward both chemotactic peptides. Taxol treatment also suppressed CXCL1- and fMLP-triggered elastase-dependent neutrophil invasion through collagen I barriers. Collectively, our results highlight de novo chemoattractant-triggered MT polymerization as key for neutrophil chemotaxis and elastase-dependent invasion but not for chemotactic neutrophil crossing of inflamed endothelial barriers., (©2019 Society for Leukocyte Biology.)

Several studies showed that hydroxyethyl starch (HES), a synthetic colloid used in volume replacement therapies, interferes with leukocyte-endothelium interactions. Although still unclear, the mechanism seems to involve the inhibition of neutrophils' integrin. With the aim to provide direct evidence of the binding of HES to neutrophils and to investigate the influence of HES on neutrophil chemotaxis, we isolated and treated the cells with different concentrations of fluorescein-conjugated HES (HES-FITC), with or without different stimuli (N-Formylmethionine-leucyl-phenylalanine, fMLP, or IL-8). HES internalization was evaluated by trypan blue quenching and ammonium chloride treatment. Chemotaxis was evaluated by under-agarose assay after pretreatment of the cells with HES or a balanced saline solution. The integrin interacting with HES was identified by using specific blocking antibodies. Our results showed that HES-FITC binds to the plasma membrane of neutrophils without being internalized. Additionally, the cell-associated fluorescence increased after stimulation of neutrophils with fMLP ( p < 0.01) but not IL-8. HES treatment impaired the chemotaxis only towards fMLP, event mainly ascribed to the inhibition of CD-11b (Mac-1 integrin) activity. Therefore, the observed effect mediated by HES should be taken into account during volume replacement therapies. Thus, HES treatment could be advantageous in clinical conditions where a low activation/recruitment of neutrophils may be beneficial, but may be harmful when unimpaired immune functions are mandatory.

A dogma of innate immunity is that neutrophils use G-protein-coupled receptors (GPCRs) for chemoattractant to chase bacteria through chemotaxis and then use phagocytic receptors coupled with tyrosine kinases to destroy opsonized bacteria via phagocytosis. Our current work showed that G-protein-coupled formyl peptide receptors (FPRs) directly mediate neutrophil phagocytosis. Mouse neutrophils lacking formyl peptide receptors (Fpr1/2 -/- ) are defective in the phagocytosis of Escherichia coli and the chemoattractant N-formyl-Met-Leu-Phe (fMLP)-coated beads. fMLP immobilized onto the surface of a bead interacts with FPRs, which trigger a Ca 2+ response and induce actin polymerization to form a phagocytic cup for engulfment of the bead. This chemoattractant GPCR/Gi signaling works independently of phagocytic receptor/tyrosine kinase signaling to promote phagocytosis. Thus, in addition to phagocytic receptor-mediated phagocytosis, neutrophils also utilize the chemoattractant GPCR/Gi signaling to mediate phagocytosis to fight against invading bacteria.

Neutrophils reach the sites of inflammation and infection in a timely manner by navigating efficiently through mechanically complex interstitial spaces, following the guidance of chemical gradients. However, our understanding of how neutrophils that follow chemical cues overcome mechanical obstacles in their path is restricted by the limitations of current experimental systems. Observations in vivo provide limited insights due to the complexity of the tissue environment. Here, we developed microfluidic devices to study the effect of progressive mechanical confinement on the migration patterns of human neutrophils toward chemical attractants. Using these devices, we identified four migration patterns: arrest, oscillation, retrotaxis, and persistent migration. The proportion of these migration patterns is different in patients receiving immunosuppressant treatments after kidney transplant, patients in critical care, and neonatal patients with infections and is distinct from that in healthy donors. The occurrence of these migration patterns is independent of the nuclear lobe number of the neutrophils and depends on the integrity of their cytoskeletal components. Our study highlights the important role of mechanical cues in moving neutrophils and suggests the mechanical constriction-induced migration patterns as potential markers for infection and inflammation., (©2018 Society for Leukocyte Biology.)

N-Formyl-Met-Leu-Phe (fMLP), a mimic of N-formyl oligopeptides that are released from bacteria, is a potent leukocyte chemotactic factor. It induces intracellular calcium ([Ca]i) transient that is important for various neutrophil biological functions, e.g., adhesion, ROS, and cytokine productions. Toll-like receptors (TLRs), an essential part of host innate immunity, regulate neutrophil activities, but their role in [Ca]i signaling is less clear. In the present study, we examined the effect of several TLR ligands, including Pam3Cys4 (TLR1/2), lipopolysaccharide (LPS, TLR4), and lipoteichoic acid (LTA, TLR2/6), on calcium signaling and on the fMLP-induced [Ca]i transients in mouse neutrophils loaded with Fura-2/AM. We found that unlike fMLP, the three TLR ligands tested did not elicit any detectable Ca flux. However, Pam3Cys4, but not LPS or LTA, markedly synergized the fMLP-induced [Ca]i transients, and had no effect on the host component keratinocyte-derived cytokine (KC)- or C5a-induced calcium flux. The effect of Pam3Cys4 on the fMLP-induced [Ca]i transients is by enhancing extracellular Ca influx, not intracellular Ca release. Surprisingly, deletion of TLR2 or MyD88 in neutrophils had no impact on the Pam3Cys4's effect, suggesting a TLR2-MyD88-independent mechanism. Finally, using the pan PKC activator and inhibitor, we demonstrated that PKC negatively regulated fMLP-induced [Ca]i transients and that inhibition of PKC did not prohibit Pam3Cys4's synergistic effect on the fMLP-induced calcium influx. In conclusion, the present study identified a novel synergistic effect of Pam3Cys4 on fMLP-induced [Ca]i transients, a process important for many neutrophil biological functions.

Neutrophils are important mediators of the innate immune defense and of the host response to a physical trauma. Because aberrant infiltration of injured sites by neutrophils was shown to cause adverse effects after trauma, we investigated how neutrophil infiltration could be modulated at the cellular level. Our data indicate that protein kinase D (PKD) is a vital regulator of neutrophil transmigration. PKD phosphorylates the Cofilin-phosphatase Slingshot-2L (SSH-2L). SSH-2L in turn dynamically regulates Cofilin activity and actin polymerization in response to a chemotactic stimulus for neutrophils, for example, fMLP. Here, we show that inhibition of PKD by two specific small molecule inhibitors results in broad, unrestricted activation of Cofilin and strongly increases the F-actin content of neutrophils even under basal conditions. This phenotype correlates with a significantly impaired neutrophil deformability as determined by optical stretcher analysis. Consequently, inhibition of PKD impaired chemotaxis as shown by reduced extravasation of neutrophils. Consequently, we demonstrate that transendothelial passage of both, neutrophil-like NB4 cells and primary PMNs recovered from a hemorrhagic shock trauma model was significantly reduced. Thus, inhibition of PKD may represent a promising modulator of the neutrophil response to trauma., (©2018 Society for Leukocyte Biology.)

Five new cembranoid-related diterpenoids, namely, flexibilisins D and E ( 1 and 2 ), secoflexibilisolides A and B ( 3 and 4 ), and flexibilisolide H ( 5 ), along with nine known compounds ( 6 ⁻ 14 ), were isolated from the soft coral Sinularia flexibilis. Their structures were established by extensive spectral analysis. Compound 3 possesses an unusual skeleton that could be biogenetically derived from cembranoids. The cytotoxicity and anti-inflammatory activities of the isolates were investigated, and the results showed that dehydrosinulariolide ( 7 ) and 11- epi -sinulariolide acetate ( 8 ) exhibited cytotoxicity toward a limited panel of cancer cell lines and 14-deoxycrassin ( 9 ) displayed anti-inflammatory activity by inhibition of superoxide anion generation and elastase release in N -formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced human neutrophils.

Six new cembranoids, cherbonolides A-E ( 1 ⁻ 5 ) and bischerbolide peroxide ( 6 ), along with one known cembranoid, isosarcophine ( 7 ), were isolated from the Formosan soft coral Sarcophyton cherbonnieri . The structures of these compounds were elucidated by detailed spectroscopic analysis and chemical methods. Compound 6 was discovered to be the first example of a molecular skeleton formed from two cembranoids connected by a peroxide group. Compounds 1 ⁻ 7 were shown to have the ability of inhibiting the production of superoxide anions and elastase release in N -formyl-methionyl-leucyl-phenyl-alanine/cytochalasin B (fMLF/CB)-induced human neutrophils.

In the vasculature, NADPH oxidase is the main contributor of reactive oxygen species (ROS) which play a key role in endothelial signalling and functions. We demonstrate that ECV304 cells express p47 phox , p67 phox , and p22 phox subunits of NADPH oxidase, as well as formyl peptide receptors 1 and 3 (FPR1/3), which are members of the GPCR family. By RT-PCR, we also detected Flt-1 and Flk-1/KDR in these cells. Stimulation of FPR1 by N-fMLP induces p47 phox phosphorylation, which is the crucial event for NADPH oxidase-dependent superoxide production. Transphosphorylation of RTKs by GPCRs is a biological mechanism through which the information exchange is amplified throughout the cell. ROS act as signalling intermediates in the transactivation mechanism. We show that N-fMLP stimulation induces the phosphorylation of cytosolic Y951, Y996, and Y1175 residues of VEGFR2, which constitute the anchoring sites for signalling molecules. These, in turn, activate PI3K/Akt and PLC- γ 1/PKC intracellular pathways. FPR1-induced ROS production plays a critical role in this cross-talk mechanism. In fact, inhibition of FPR1 and/or NADPH oxidase functions prevents VEGFR2 transactivation and the triggering of the downstream signalling cascades. N-fMLP stimulation also ameliorates cellular migration and capillary-like network formation ability of ECV304 cells.

Three new polyoxygenated steroids, michosterols A-C ( 1 - 3 ), and four known compounds ( 4 - 7 ) were isolated from the ethyl acetate (EtOAc) extract of the soft coral Lobophytum michaelae , collected off the coast of Taitung. The structures of the new compounds were elucidated on the basis of spectroscopic analyses and comparison of the nuclear magnetic resonance (NMR) data with related steroids. The cytotoxicity of compounds 1 - 3 against the proliferation of a limited panel of cancer cell lines was assayed. Compound 1 was found to display moderate cytotoxicity against adenocarcinomic human alveolar basal epithelial (A549) cancer cells. It also exhibited potent anti-inflammatory activity by suppressing superoxide anion generation and elastase release in N -formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-stimulated human neutrophils. Furthermore, 3 could effectively inhibit elastase release, as well., Competing Interests: The authors declare no conflicts of interest.

Neutrophils are key cellular components of the innate immune response and characteristically migrate from the blood towards and throughout tissues. Their migratory process is complex, guided by multiple chemoattractants released from injured tissues and microbes. How neutrophils integrate the various signals in the tissue microenvironment and mount effective responses is not fully understood. Here, we employed microfluidic mazes that replicate features of interstitial spaces and chemoattractant gradients within tissues to analyze the migration patterns of human neutrophils. We find that neutrophils respond to LTB4 and fMLF gradients with highly directional migration patterns and converge towards the source of chemoattractant. We named this directed migration pattern convergent. Moreover, neutrophils respond to gradients of C5a and IL-8 with a low-directionality migration pattern and disperse within mazes. We named this alternative migration pattern divergent. Inhibitors of MAP kinase and PI-3 kinase signaling pathways do not alter either convergent or divergent migration patterns, but reduce the number of responding neutrophils. Overlapping gradients of chemoattractants conserve the convergent and divergent migration patterns corresponding to each chemoattractant and have additive effects on the number of neutrophils migrating. These results suggest that convergent and divergent neutrophil migration-patterns are the result of simultaneous activation of multiple signaling pathways.

New lobane-based diterpenoids lobovarols A-D ( 1 - 4 ) and a prenyleudesmane-type diterpenoid lobovarol E ( 5 ) along with seven known related diterpenoids ( 6 - 12 ) were isolated from the ethyl acetate extract of a Taiwanese soft coral Lobophytum varium . Their structures were identified on the basis of multiple spectroscopic analyses and spectral comparison. The absolute configuration at C-16 of the known compound 11 is reported herein for the first time. The anti-inflammatory activities of compounds 1 - 12 were assessed by measuring their inhibitory effect on N -formyl-methionyl-leucyl-phenyl-alanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release in human neutrophils. Metabolites 2 , 5 , and 11 were found to show moderate inhibitory activity on the generation of superoxide anion, while compounds 5 , 8 , 11 , and 12 could effectively suppress elastase release in fMLP/CB-stimulated human neutrophil cells at 10 μM. All of the isolated diterpenoids did not exhibit cytotoxic activity (IC 50 > 50 μM) towards a limited panel of cancer cell lines., Competing Interests: The authors declare no conflict of interest.

The niacin receptor HCA2 is implicated in controlling inflammatory host responses with yet poorly understood mechanistic basis. We previously reported that HCA2 in A431 epithelial cells transduced Gβγ-protein kinase C- and Gβγ-metalloproteinase/EGFR-dependent MAPK/ERK signaling cascades. Here, we investigated the role of HCA2 in macrophage-mediated inflammation and the underlying mechanisms. We found that proinflammatory stimulants LPS, IL-6 and IL-1β up-regulated the expression of HCA2 on macrophages. Niacin significantly inhibited macrophage chemotaxis in response to chemoattractants fMLF and CCL2 by disrupting polarized distribution of F-actin and Gβ protein. Niacin showed a selected additive effect on chemoattractant-induced activation of ERK1/2, JNK and PI3K pathways, but only the MEK inhibitor UO126 reduced niacin-mediated inhibition of macrophage chemotaxis, while activation of ERK1/2 by EGF alone did not inhibit fMLF-mediated migration of HEK293T cells co-expressing HCA2 and fMLF receptor FPR1. In addition, niacin induced heterologous desensitization and internalization of FPR1. Furthermore, niacin rescued mice from septic shock by diminishing inflammatory symptoms and the effect was abrogated in HCA2 -/- mice. These results suggest that Gβγ/PKC-dependent ERK1/2 activation and heterologous desensitization of chemoattractant receptors are involved in the inhibition of chemoattractant-induced migration of macrophages by niacin. Thus, HCA2 plays a critical role in host protection against pro-inflammatory insults., Competing Interests: The authors declare no competing financial interests.

β-Arrestins have emerged as key regulators of cytoskeletal rearrangement that are required for directed cell migration. Whereas it is known that β-arrestins are required for formyl-Met-Leu-Phe receptor (FPR) recycling, less is known about their role in regulating FPR-mediated neutrophil chemotaxis. Here, we show that β-arrestin 1 (ArrB1) coaccumulated with F-actin within the leading edge of neutrophil-like HL-60 cells during chemotaxis, and its knockdown resulted in markedly reduced migration within fMLP gradients. The small GTPase Ras-related protein 2 (Rap2) was found to bind ArrB1 under resting conditions but dissociated upon fMLP stimulation. The FPR-dependent activation of Rap2 required ArrB1 but was independent of Gα i activity. Significantly, depletion of either ArrB1 or Rap2 resulted in reduced chemotaxis and defects in cellular repolarization within fMLP gradients. These data strongly suggest a model in which FPR is able to direct ArrB1 and other bound proteins that are required for lamellipodial extension to the leading edge in migrating neutrophils, thereby orientating and directing cell migration., (© Society for Leukocyte Biology.)

Neutrophils play an essential role in innate immunity due to their ability to migrate into infected tissues and kill microbes with bactericides located in their secretory granules. Neutrophil transmigration and degranulation are tightly regulated by actin cytoskeleton. Invading pathogens produce alkaloids that cause the depolymerization of actin, such as the mold alkaloid cytochalasin D. We studied the effect of cytochalasin D on the morphology and secretion of fMLP-, LPS-, or PMA-stimulated human neutrophils upon adhesion to fibronectin. Electron microscopy showed that the morphology of the neutrophils adherent to fibronectin in the presence of various stimuli differed. But in the presence of cytochalasin D, all stimulated neutrophils exhibited a uniform nonspread shape and developed thread-like membrane tubulovesicular extensions (cytonemes) measuring 200 nm in diameter. Simultaneous detection of neutrophil secretory products by mass spectrometry showed that all tested stimuli caused the secretion of MMP-9, a key enzyme in the neutrophil migration. Cytochalasin D impaired the MMP-9 secretion but initiated the release of cathepsin G and other granular bactericides, proinflammatory agents. The release of bactericides apparently occurs through the formation, shedding, and lysis of cytonemes. The production of alkaloids which modify neutrophil responses to stimulation via actin depolymerization may be part of the strategy of pathogen invasion.

Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, plays critical roles in phagocytic host defense. However, how activated neutrophils regulate CFTR channel distribution subcellularly is not well defined. To investigate, we tested multiple Abs against different CFTR domains, to examine CFTR expression in human peripheral blood neutrophils by flow cytometry. The data confirmed that resting neutrophils had pronounced CFTR expression. Activation of neutrophils with soluble or particulate agonists did not significantly increase CFTR expression level, but induced CFTR redistribution to cell surface. Such CFTR mobilization correlated with cell-surface recruitment of formyl-peptide receptor during secretory vesicle exocytosis. Intriguingly, neutrophils from patients with ΔF508-CF, despite expression of the mutant CFTR, showed little cell-surface mobilization upon stimulation. Although normal neutrophils effectively targeted CFTR to their phagosomes, ΔF508-CF neutrophils had impairment in that process, resulting in deficient hypochlorous acid production. Taken together, activated neutrophils regulate CFTR distribution by targeting this chloride channel to the subcellular sites of activation, and ΔF508-CF neutrophils fail to achieve such targeting, thus undermining their host defense function., (© Society for Leukocyte Biology.)

Two different immune recognition systems have evolved in parallel to recognize peptides starting with an N-formylated methionine, and recognition similarities/differences between these two systems have been investigated. A number of peptides earlier characterized in relation to the H2-M3 complex that presents N-formylated peptides to cytotoxic T cells, have been characterized in relation to the formyl peptide receptors expressed by phagocytic neutrophils in both men (FPRs) and mice (Fprs). FPR1/Fpr1 was identified as the preferred receptor for all fMet-containing peptides examined, but there was no direct correlation between H2-M3 binding and the neutrophil activation potencies. Similarly, there was no direct correlation between the activities induced by the different peptides in human and mouse neutrophils, respectively. The formyl group was important in both H2-M3 binding and FPR activation, but FPR2 was the preferred receptor for the non-formylated peptide. The structural requirements differed between the H2-M3 and FPR/Fpr recognition systems and these data suggest that the two recognition systems have different evolutionary traits., Competing Interests: The authors have declared that no competing interests exist.

Neutrophils are the most abundant leukocytes in peripheral blood and play critical a role in bacterial infection, tumor immunity and wound repair. Clarifying the process of neutrophil chemotaxis to target sites of immune activity has been a focus of increased interest within the past decade. In bacterial infectious foci, neutrophils migrate toward the bacterial-derived chemoattractant N-formyl-Met-Leu-Phe (fMLP) and ignore other intermediary chemoattractants to arrive at the area of infection. Using an under agarose chemotaxis assay, we observed that the bacterial fMLP-induced neutrophil chemotaxis signal overrode interleukin 8 (IL-8)- and leukotriene B4 (LTB4)-induced chemotaxis signals. Moreover, in the presence of bacterial lipopolysaccharide (LPS), the fMLP-induced hierarchical chemotaxis signal was enhanced. Further studies revealed that LPS increased the membrane expression of the fMLP receptor, formyl peptide receptor 1 (FPR1). However, expression levels of the membrane receptors for IL-8 and LTB4 were decreased by LPS administration. A human Phospho-mitogen-activated protein kinase (MAPK) proteome array showed that the p38 pathway was significantly activated by LPS stimulation. Moreover, p38 was responsible for the altered expression of neutrophil membrane chemoattractant receptors. Inhibition of neutrophil p38 restored LPS-improved hierarchical chemotaxis. Taken together, these data indicate that endotoxin promotes neutrophil hierarchical chemotaxis via the p38-membrane receptor pathway.

Progressive lung disease with early onset is the main cause of mortality and morbidity in cystic fibrosis patients. Here we report a reduction of sphingosine-1-phosphate (S1P) in the lung of unchallenged Cftr tm1EUR F508del CFTR mutant mice. This correlates with enhanced infiltration by inducible nitric oxide synthase (iNOS)-expressing granulocytes, B cells, and T cells. Furthermore, the ratio of macrophage-derived dendritic cells (MoDC) to conventional dendritic cells (cDC) is higher in mutant mouse lung, consistent with unprovoked inflammation. Oral application of a S1P lyase inhibitor (LX2931) increases S1P levels in mutant mouse tissues. This normalizes the lung MoDC/cDC ratio and reduces B and T cell counts. Lung granulocytes are enhanced, but iNOS expression is reduced in this population. Although lung LyC6+ monocytes are enhanced by LX2931, they apparently do not differentiate to MoDC and macrophages. After challenge with bacterial toxins (LPS-fMLP) we observe enhanced levels of proinflammatory cytokines TNF-α, KC, IFNγ, and IL-12 and the inducible mucin MUC5AC in mutant mouse lung, evidence of deficient resolution of inflammation. LX2931 does not prevent transient inflammation or goblet cell hyperplasia after challenge, but it reduces MUC5AC and proinflammatory cytokine levels toward normal values. We conclude that lung pathology in homozygous mice expressing murine F508del CFTR, which represents the most frequent mutation in CF patients, is characterized by abnormal behavior of infiltrating myeloid cells and delayed resolution of induced inflammation. This phenotype can be partially corrected by a S1P lyase inhibitor, providing a rationale for therapeutic targeting of the S1P signaling pathway in CF patients., (Copyright © 2016 the American Physiological Society.)

A key step in neutrophil-mediated tissue damage is the migration of activated neutrophils across the vascular endothelium. Previously, we identified protein kinase C δ as a critical regulator of neutrophil migration in sepsis but did not identify specific steps in migration. In this study, we used our novel biomimetic microfluidic assay to delineate systematically the mechanism by which protein kinase C δ regulates individual steps in human neutrophil-endothelial interaction during inflammation. The biomimetic microfluidic assay includes a network of vascular channels, produced from in vivo images connected to a tissue compartment through a porous barrier. HUVECs cultured in vascular channels formed a complete lumen under physiologic shear flow. HUVECs were pretreated with TNF-α ± a protein kinase C δ inhibitor, and the tissue compartment was filled with a chemoattractant (fMLP or IL-8). Under physiologic shear flow, the role of protein kinase C δ on spatial and temporal neutrophil adherence/migration was quantified. Protein kinase C δ inhibition significantly reduced neutrophil adhesion in response to fMLP and IL-8 only under low shear rate and near bifurcations. Protein kinase C δ inhibition also decreased adherence to nonactivated HUVECs in response to fMLP or IL-8. Protein kinase C δ inhibition reduced neutrophil migration into the tissue compartment in response to fMLP and to a lesser degree, to IL-8. Antibody-coated microparticles demonstrated that protein kinase C δ inhibition down-regulated E-selectin and ICAM-1 but not VCAM-1 expression. With the use of a physiologically relevant in vitro model system, we demonstrate that protein kinase C δ plays an important role in the regulation of neutrophil adherence/migration during inflammation and identifies key steps regulated by protein kinase C δ in neutrophil-endothelial interactions., (© Society for Leukocyte Biology.)

Leukocyte crawling and transendothelial migration (TEM) are potentiated by shear stress caused by blood flow. The mechanism that couples shear stress to migration has not been fully elucidated. We found that mice lacking GEF-H1 (GEF-H1 -/- ), a RhoA-specific guanine nucleotide exchange factor (GEF), displayed limited migration and recruitment of neutrophils into inflamed tissues. GEF-H1 -/- leukocytes were deficient in in vivo crawling and TEM in the postcapillary venules. We demonstrated that although GEF-H1 deficiency had little impact on the migratory properties of neutrophils under static conditions, shear stress triggered GEF-H1-dependent spreading and crawling of neutrophils and relocalization of GEF-H1 to flotillin-2-rich uropods. Our results identify GEF-H1 as a component of the shear stress response machinery in neutrophils required for a fully competent immune response to bacterial infection., (© 2016 Fine et al.)

Aminopeptidase N (CD13) is a widely expressed cell surface metallopeptidase involved in the migration of cancer and endothelial cells. Apart from our demonstration that CD13 modulates the efficacy of tumor necrosis factor-α-induced apoptosis in neutrophils, no other function for CD13 has been ascribed in this cell. We hypothesized that CD13 may be involved in neutrophil migration and/or homotypic aggregation. Using purified human blood neutrophils we confirmed the expression of CD13 on neutrophils and its up-regulation by pro-inflammatory agonists. However, using the anti-CD13 monoclonal antibody WM-15 and the aminopeptidase enzymatic inhibitor bestatin we were unable to demonstrate any direct involvement of CD13 in neutrophil polarisation or chemotaxis. In contrast, IL-8-mediated neutrophil migration in type I collagen gels was significantly impaired by the anti-CD13 monoclonal antibodies WM-15 and MY7. Notably, these antibodies also induced significant homotypic aggregation of neutrophils, which was dependent on CD13 cross-linking and was attenuated by phosphoinositide 3-kinase and extracellular signal-related kinase 1/2 inhibition. Live imaging demonstrated that in WM-15-treated neutrophils, where homotypic aggregation was evident, the number of cells entering IL-8 impregnated collagen I gels was significantly reduced. These data reveal a novel role for CD13 in inducing homotypic aggregation in neutrophils, which results in a transmigration deficiency; this mechanism may be relevant to neutrophil micro-aggregation in vivo.

Monocyte migration requires the dynamic redistribution of integrins through a regulated endo-exocytosis cycle, but the complex molecular mechanisms underlying this process have not been fully elucidated. Glia maturation factor-γ (GMFG), a novel regulator of the Arp2/3 complex, has been shown to regulate directional migration of neutrophils and T-lymphocytes. In this study, we explored the important role of GMFG in monocyte chemotaxis, adhesion, and β1-integrin turnover. We found that knockdown of GMFG in monocytes resulted in impaired chemotactic migration toward formyl-Met-Leu-Phe (fMLP) and stromal cell-derived factor 1α (SDF-1α) as well as decreased α5β1-integrin-mediated chemoattractant-stimulated adhesion. These GMFG knockdown impaired effects could be reversed by cotransfection of GFP-tagged full-length GMFG. GMFG knockdown cells reduced the cell surface and total protein levels of α5β1-integrin and increased its degradation. Importantly, we demonstrate that GMFG mediates the ubiquitination of β1-integrin through knockdown or overexpression of GMFG. Moreover, GMFG knockdown retarded the efficient recycling of β1-integrin back to the plasma membrane following normal endocytosis of α5β1-integrin, suggesting that the involvement of GMFG in maintaining α5β1-integrin stability may occur in part by preventing ubiquitin-mediated degradation and promoting β1-integrin recycling. Furthermore, we observed that GMFG interacted with syntaxin 4 (STX4) and syntaxin-binding protein 4 (STXBP4); however, only knockdown of STXBP4, but not STX4, reduced monocyte migration and decreased β1-integrin cell surface expression. Knockdown of STXBP4 also substantially inhibited β1-integrin recycling in human monocytes. These results indicate that the effects of GMFG on monocyte migration and adhesion probably occur through preventing ubiquitin-mediated proteasome degradation of α5β1-integrin and facilitating effective β1-integrin recycling back to the plasma membrane., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mechanobiology relates cellular processes to mechanical signals, such as determining the effect of variations in matrix stiffness with cell tractions. Cell traction recorded via traction force microscopy (TFM) commonly takes place on materials such as polyacrylamide- and polyethylene glycol-based gels. Such experiments remain limited in physiological relevance because cells natively migrate within complex tissue microenvironments that are spatially heterogeneous and hierarchical. Yet, TFM requires determination of the matrix constitutive law (stress-strain relationship), which is not always readily available. In addition, the currently achievable displacement resolution limits the accuracy of TFM for relatively small cells. To overcome these limitations, and increase the physiological relevance of in vitro experimental design, we present a new approach and a set of associated biomechanical signatures that are based purely on measurements of the matrix's displacements without requiring any knowledge of its constitutive laws. We show that our mean deformation metrics (MDM) approach can provide significant biophysical information without the need to explicitly determine cell tractions. In the process of demonstrating the use of our MDM approach, we succeeded in expanding the capability of our displacement measurement technique such that it can now measure the 3D deformations around relatively small cells (∼10 micrometers), such as neutrophils. Furthermore, we also report previously unseen deformation patterns generated by motile neutrophils in 3D collagen gels.

Three new triterpenoids; namely 28,28,30-trihydroxylupeol (1); 3,21,21,26-tetrahydroxy-lanostanoic acid (2) and dehydroxybetulinic acid (3) and seven known compounds; i.e., taraxerone (4); taraxerol (5); ethyl palmitate (6); herniarin (7); stigmasterol (8); ursolic acid (9) and acetyl ursolic acid (10) were isolated from the stem of Ficus aurantiaca Griff. The structures of the compounds were established by spectroscopic techniques. The compounds were evaluated for their inhibitory effects on polymorphonuclear leukocyte (PMN) chemotaxis by using the Boyden chamber technique and on human whole blood and neutrophil reactive oxygen species (ROS) production by using a luminol-based chemiluminescence assay. Among the compounds tested, compounds 1-4, 6 and 9 exhibited strong inhibition of PMN migration towards the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) with IC50 values of 6.8; 2.8; 2.5; 4.1; 3.7 and 3.6 μM, respectively, comparable to that of the positive control ibuprofen (6.7 μM). Compounds 2-4, 6, 7 and 9 exhibited strong inhibition of ROS production of PMNs with IC50 values of 0.9; 0.9; 1.3; 1.1; 0.5 and 0.8 μM, respectively, which were lower than that of aspirin (9.4 μM). The bioactive compounds might be potential lead molecules for the development of new immunomodulatory agents to modulate the innate immune response of phagocytes.

The leaves of Perilla frutescens (L.) Britt. have been traditionally used as an herbal medicine in East Asian countries to treat a variety diseases. In this present study, we investigated the inhibitory effects of P. frutescens extract (PFE) on N-formyl-Met-Leu-Phe (fMLF)-stimulated human neutrophils and the underlying mechanisms. PFE (1, 3, and 10 μg/ml) inhibited superoxide anion production, elastase release, reactive oxygen species formation, CD11b expression, and cell migration in fMLF-activated human neutrophils in dose-dependent manners. PFE inhibited fMLF-induced phosphorylation of the Src family kinases (SFKs), Src (Tyr416) and Lyn (Tyr396), and reduced their enzymatic activities. Both PFE and PP2 (a selective inhibitor of SFKs) reduced the phosphorylation of Burton's tyrosine kinases (Tyr223) and Vav (Tyr174) in fMLF-activated human neutrophils. Additionally, PFE decreased intracellular Ca(2+) levels ([Ca(2+)]i), whereas PP2 prolonged the time required for [Ca(2+)]i to return to its basal level. Our findings indicated that PFE effectively regulated the inflammatory activities of fMLF-activated human neutrophils. The anti-inflammatory effects of PFE on activated human neutrophils were mediated through two independent signaling pathways involving SFKs (Src and Lyn) and mobilization of intracellular Ca(2+).

A new limonoid, swietemacrophin (1), was isolated from the seeds of Swietenia macrophylla, together with five known compounds 2-6. The structure of 1 was determined through extensive 1D/2D-NMR and mass-spectrometric analyses. Swietemacrophin (1), humilinolide F (2), 3,6-O,O-diacetylswietenolide (3), 3-O-tigloylswietenolide (4), and swietemahonin E (5) exhibited inhibition (IC50 values≤45.44 μM) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Compounds 1, 4, 5, and swietenine (6) showed potent inhibition with IC50 values≤36.32 μM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation.

Regulation of neutrophil activity is critical for immune evasion among extracellular pathogens, yet the mechanisms by which many bacteria disrupt phagocyte function remain unclear. Here, we have shown that the respiratory pathogen Streptococcus pneumoniae disables neutrophils by exploiting molecular mimicry to degrade platelet-activating factor (PAF), a host-derived inflammatory phospholipid. Using mass spectrometry and murine upper airway infection models, we demonstrated that phosphorylcholine (ChoP) moieties that are shared by PAF and the bacterial cell wall allow S. pneumoniae to leverage a ChoP-remodeling enzyme (Pce) to remove PAF from the airway. S. pneumoniae-mediated PAF deprivation impaired viability, activation, and bactericidal capacity among responding neutrophils. In the absence of Pce, neutrophils rapidly cleared S. pneumoniae from the airway and impeded invasive disease and transmission between mice. Abrogation of PAF signaling rendered Pce dispensable for S. pneumoniae persistence, reinforcing that this enzyme deprives neutrophils of essential PAF-mediated stimulation. Accordingly, exogenous activation of neutrophils overwhelmed Pce-mediated phagocyte disruption. Haemophilus influenzae also uses an enzyme, GlpQ, to hydrolyze ChoP and subvert PAF function, suggesting that mimicry-driven immune evasion is a common paradigm among respiratory pathogens. These results identify a mechanism by which shared molecular structures enable microbial enzymes to subvert host lipid signaling, suppress inflammation, and ensure bacterial persistence at the mucosa.

Formylated peptides are chemotactic agents generated by pathogens. The most relevant peptide is fMLF (formyl-Met-Leu-Phe) which participates in several immune functions, such as chemotaxis, phagocytosis, cytokine release and generation of reactive oxygen species. In macrophages fMLF-dependent responses are dependent on both, an increase in intracellular calcium concentration and on a hyperpolarization of the membrane potential. However, the molecular entity underlying this hyperpolarization remains unknown and it is not clear whether changes in membrane potential are linked to the increase in intracellular Ca2+. In this study, differentiated U937 cells, as a macrophage-like cell model, was used to characterize the fMLF response using electrophysiological and Ca2+ imaging techniques. We demonstrate by means of pharmacological and molecular biology tools that fMLF induces a Ca2+-dependent hyperpolarization via activation of the K+ channel KCa3.1 and thus, enhancing fMLF-induced intracellular Ca2+ increase through an amplification of the driving force for Ca2+ entry. Consequently, enhanced Ca2+ influx would in turn lengthen the hyperpolarization, operating as a positive feedback mechanism for fMLF-induced Ca2+ signaling.

In human pathological conditions, the acidification of local environment is a frequent feature, such as tumor and inflammation. As the pH of microenvironment alters, the functions of immune cells are about to change. It makes the extracellular acidification a key modulator of innate immunity. Here we detected the impact of extracellular acidification on neutrophil apoptosis and functions, including cell death, respiratory burst, migration and phagocytosis. As a result, we found that under the acid environment, neutrophil apoptosis delayed, respiratory burst inhibited, polarization augmented, chemotaxis differed, endocytosis enhanced and bacteria killing suppressed. These findings suggested that extracellular acidification acts as a key regulator of neutrophil apoptosis and functions.

The undesirable respiratory burst in neutrophils can lead to inflammation and tissue damage. This study investigates the effect and the underlying mechanism of 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol (honokiol), a lignan extracted from the stem bark of Magnolia officinalis Rehd. et Wils (Magnoliaceae), on N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced respiratory burst in human neutrophils. Signaling pathways regulated by honokiol which modulate fMLP-induced respiratory burst and cathepsin G release were evaluated by phosphorylation of Src family kinase induced by fMLP, Src family kinases activities and by immunoblotting analysis of the downstream targets of Src kinase. Briefly, honokiol inhibited fMLP-induced superoxide anion production (IC50 = 9.80 ± 0.21 μM, n = 4), cathepsin G release (IC50 = 14.23 ± 1.43 μM, n = 4) and migration (IC50 = 5.69 ± 1.51 μM, n = 4) in a concentration dependent manner. Further, honokiol specifically suppresses fMLP-induced Lyn (a member of the Src kinase family) phosphorylation, by inhibiting Lyn kinase activity. Consequently, honokiol attenuated the downstream targets of Lyn kinase, such as Tec translocation from the cytosol to the inner leaflet of the plasma membrane, phosphorylation of AKT, P38, PLCγ2, protein kinase C and membrane localization of p47(phox). On the other hand, fMLP-induced phosphorylation of Hck, Fgr kinase activity (other members of Src kinase), downstream phosphorylation of Vav1 and extracellular signal-regulated kinase remained unaffected. In addition, honokiol neither inhibited NADPH oxidase activity nor increased cyclic AMP levels. Honokiol is not a competitive or allosteric antagonist of fMLP. In conclusion, honokiol specifically modulates fMLP-mediated neutrophil activation by inhibiting Lyn activation which subsequently interferes with the activation of PLCγ2, AKT, p38, protein kinase C, and p47(phox)., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Purpose: In the closed-eye environment (during sleep), there is an influx of neutrophils into the tear film, and the phenotype of these cells has yet to be characterized. This study was conducted to investigate the response of tear-film neutrophils to inflammatory stimuli., Methods: Immediately upon awakening, cells from healthy participants (n = 12) were collected using a gentle eye-wash with PBS. Tear-film neutrophils were counted and cell viability was determined. Neutrophils were also isolated from blood by density-gradient centrifugation. Tear-film and blood-isolated neutrophils were stimulated with phorbol myristate acetate (PMA), lipopolysaccharide (LPS), or N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Changes in the expression of macrophage-1 antigen, intercellular adhesion molecule-1 (ICAM-1), CD66b (a degranulation membrane marker), C3aR (complement C3a receptor), CD45 (leukocyte common antigen) as well as reactive oxygen species (using dichlorodihydro-fluorescein diacetate) were characterized by flow cytometry., Results: Hundreds of thousands of leukocytes were collected upon awakening. Tear-film neutrophils were alive as shown by trypan blue and propidium iodide (PI) exclusion. While tear-film neutrophils were able to mount an oxidative response, stimulation with LPS, PMA, or fMLP did not induce receptor upregulation. This lack of response to stimulus with tear-film neutrophils was significantly different from that of blood-isolated neutrophils. Incubation in the presence of tear film proteins did not affect the tear-film neutrophil response to stimuli., Conclusions: Our results indicate that while tear-film neutrophils are alive, they do not respond to inflammatory stimuli in the same manner as blood-isolated neutrophils. This refractory phenotype may be due to exposure to anti-inflammatory factors present in the tear film.

N-formyl peptide receptors (FPR1, FPR2 and FPR3) are involved in innate immunity, inflammation and cancer. FPR expression, initially described in immune cells, was later observed in non-hematopoietic cell populations and tissues. Several studies suggested a role for FPRs in the progression of various tumor histotypes, including gastric cancer (GC), for which a positive association with a specific FPR1 polymorphism has recently been described. We previously showed that FPRs are expressed on gastric epithelium and are required for wound repair and restitution of barrier integrity. Here we assess the role of FPRs in GC. We characterized the functions of FPRs in GC epithelial cells (MKN28, AGS and MKN45) cultured in vitro by assessing migration, proliferation, resistance to apoptosis and activation of the epithelial-to-mesenchymal transition. Activation of each FPR induced the epithelial-to-mesenchymal transition, proliferation, resistance to apoptosis and migration of GC cells in culture. Blocking compounds or RNA interference of each FPR reverted these effects. We also defined the in vivo tumorigenic potential of GC epithelial cells silenced for FPRs by xenograft experiments in immunocompromised mice. Interestingly, FPR1 silencing in GC cells (shFPR1) significantly enhanced xenograft growth with respect to shCTR, shFPR2 and shFPR3 xenografts, because of augmented vessel density and cell proliferation. Accordingly, HIF-1α and VEGF mRNA levels were higher in shFPR1 xenografts than in controls. Moreover, the in vitro production of proangiogenic factors in response to FPR2/3 agonists (WKYMVm, LL-37, uPA, uPAR84-95, AnxA1) or to other proinflammatory mediators (IL-1α) was higher in shFPR1 GC cells than in shCTR, shFPR2 and shFPR3 cells, suggesting that FPR1 functions as an inhibitor of CG angiogenesis. Thus, we propose that FPR1 stimulation may represent a novel therapeutic approach to counteract tumor angiogenesis.

The present study aimed to determine different peripheral blood neutrophil functions in 18 morbidly obese subjects with body mass index (BMI) ranging between 35 and 69 kg/m(2) in parallel with age- and gender-matched lean controls. Peripheral blood neutrophil functions of obese subjects and matched lean controls were determined. Neutrophils of obese subjects showed significant elevation of the release of basal superoxides (P < 0.0001), formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated superoxides (P < 0.0001) and opsonized zymosan (OZ)-stimulated superoxides (P < 0.045) compared with lean controls. Interestingly, there were no differences in phorbol myristate acetate (PMA)-stimulated superoxide production by neutrophils of the obese subjects and controls. There was also a significant elevation of chemotactic (P < 0.0003) and random (P < 0.0001) migration of neutrophils from obese subjects compared with lean controls. Phagocytosis, CD11b surface expression and adherence of neutrophils from obese subjects were not significantly different from those of the lean controls. The elevated superoxide production and chemotactic activity, together with the normal phagocytosis and adherence, suggest that neutrophils from obese subjects are primed and have the capability to combat infections. However, neutrophils in the priming state may participate in the pathogenesis of obesity-related diseases., (© 2015 British Society for Immunology.)