Neutrophil Motility Research Articles

MLK3 regulates fMLP-stimulated neutrophil motility

IntroductionMixed lineage kinase 3 (MLK3) is part of the intracellular regulatory system that connects extracellular cytokine or mitogen signals received through G-protein coupled receptors to changes in gene expression. MLK3 activation stimulates motility of epithelial cells and epithelial-derived tumor cells, but its role in mediating the migration of other cell types remains unknown. Since neutrophils play a crucial role in innate immunity and contribute to the pathogenesis of several diseases, we therefore examined whether MLK3 might regulate the motility of mouse neutrophils responding to a chemotactic stimulus, the model bacterial chemoattractant fMLP. MethodsThe expression of Mlk3 in mouse neutrophils was determined by immunocytochemistry and by RT-PCR. In vitro chemotaxis in a gradient of fMLP, fMLP-stimulated random motility, fMLP-stimulated F-actin formation were measured by direct microscopic observation using neutrophils pre-treated with a novel small molecule inhibitor of MLK3 (URMC099) or neutrophils obtained from Mlk3−/− mice. In vivo effects of MLK3 inhibition were measured by counting the fMLP-induced accumulation of neutrophils in the peritoneum following pre-treatment with URMC099 in wild-type C57Bl/6 or mutant Mlk3−/− mice. ResultsThe expression of Mlk3 mRNA and protein was observed in neutrophils purified from wild-type C57Bl/6 mice but not in neutrophils from mutant Mlk3−/− mice. Chemotaxis by wild-type neutrophils induced by a gradient of fMLP was reduced by pre-treatment with URMC099. Neutrophils from C57Bl/6 mice pretreated with URMC099 and neutrophils from Mlk3−/− mice moved far less upon fMLP-stimulation and did not form F-actin as readily as untreated neutrophils from C57Bl/6 controls. In vivo recruitment of neutrophils into the peritoneum by fMLP was significantly reduced in wild-type mice treated with URMC099, as well as in untreated Mlk3−/− mice—thereby confirming the role of MLK3 in neutrophil migration. ConclusionsMlk3 mRNA is expressed in murine neutrophils. Genetic or pharmacologic inhibition of MLK3 blocks fMLP-mediated motility of neutrophils both in vitro and in vivo, suggesting that MLK3 may be a therapeutic target in human diseases characterized by exuberant neutrophil migration.

Heat Shock Modulates Neutrophil Motility in Zebrafish

Heat shock is a routine method used for inducible gene expression in animal models including zebrafish. Environmental temperature plays an important role in the immune system and infection progression of ectotherms. In this study, we analyzed the impact of short-term heat shock on neutrophil function using zebrafish (Danio rerio) as an animal model. Short-term heat shock decreased neutrophil recruitment to localized Streptococcus iniae infection and tail fin wounding. Heat shock also increased random neutrophil motility transiently and increased the number of circulating neutrophils. With the use of the translating ribosome affinity purification (TRAP) method for RNA isolation from specific cell types such as neutrophils, macrophages and epithelial cells, we found that heat shock induced the immediate expression of heat shock protein 70 (hsp70) and a prolonged expression of heat shock protein 27 (hsp27). Heat shock also induced cell stress as detected by the splicing of X-box binding protein 1 (xbp1) mRNA, a marker for endoplasmic reticulum (ER) stress. Exogenous expression of Hsp70, Hsp27 and spliced Xbp1 in neutrophils or epithelial cells did not reproduce the heat shock induced effects on neutrophil recruitment. The effect of heat shock on neutrophils is likely due to a combination of complex changes, including, but not limited to changes in gene expression. Our results indicate that routine heat shock can alter neutrophil function in zebrafish. The findings suggest that caution should be taken when employing a heat shock-dependent inducible system to study the innate immune response.

Mechanisms affecting neutrophil migration capacity in breast cancer patients before and after chemotherapy

To investigate the mechanisms affecting neutrophil migration capacity in breast cancer patients before and after chemotherapy. Peripheral venous blood was collected at the time of diagnosis and immediately prior to the 4th cycle of an anthracycline-based chemotherapy regimen for patients diagnosed with different stages of breast cancer (n=30), for experimental assays. Blood samples were also collected from a healthy control group (n=17). IL-8 serum concentrations were higher in the patient group than in the control group (p=0.02), and chemotherapy did not further affect this increase. Levels of TNF-α, IL-6, and IL-10 did not differ between controls and patients, or in relation to chemotherapy. Serum levels of nitric oxide (NO) metabolites were elevated following chemotherapy compared to levels detected prior to treatment (p=0.01). When the supernatants of lipopolysaccharide-stimulated mononuclear cells and neutrophils obtained from the patients were assayed for levels of nitrite, these levels were significantly higher and unchanged, respectively, compared with controls. Expression levels of the chemokine receptors, CXCR1 and CXCR2, were significantly reduced in patients compared to controls, and chemotherapy did not further affect these differences. Furthermore, filamentous actin content for IL-8-activated neutrophils was reduced with chemotherapy (median 8.85; range 3.38-13.43) compared to the content detected prior to treatment (median 9.23; range 2.86-22.16) (p=0.001). Elevated systemic levels of IL-8 and NO, desensitization to CXCR activation, and reduction in actin polymerization may affect neutrophil motility in patients before and after chemotherapy.

S96 Simvastatin as an adjuvant therapy for infection and sepsis–in-vitro and in-vivo studies suggest pre-emptive / early therapy in the elderly

Ageing is associated with increased episodes of sepsis and poorer outcomes. Statins are associated with improved outcomes during infection. We aimed to characterise the impact of age and acute severe...

Calpain-1 Inhibitors for Selective Treatment of Rheumatoid Arthritis: What is the Future?

Effective small-molecule treatment of inflammatory diseases remains an unmet need in medicine. Current treatments are either limited in effectiveness or invasive. The latest biologics prevent influx of inflammatory cells to damaged tissue. Calpain-1 is a calcium-activated cysteine protease that plays an important role in neutrophil motility. It is, therefore, a potential target for intervention in inflammatory disease. Many inhibitors of calpains have been developed but most are unselective and so unsuitable for drug use. However, recent series of α-mercaptoacrylate inhibitors target regulatory domains of calpain-1 and are much more specific. These compounds are effective in impairing the cell spreading mechanism of neutrophils in vitro and raise the possibility of treating rheumatoid arthritis with a pill; however, challenges still remain. Improved bioavailability is needed and solution of their precise mode of action should prompt the development of specific calpain-1 screens for novel classes of inhibitors.

DAPK2 positively regulates motility of neutrophils and eosinophils in response to intermediary chemoattractants

The tight regulation of granulocyte chemotaxis is crucial for initiation and resolution of inflammation. Here, we show that DAPK2, a Ca(2+)/CaM-sensitive serine/threonine kinase known to modulate cell death in various cell types, is a novel regulator of migration in granulocytes. We demonstrate that human neutrophils and eosinophils express DAPK2 but unlike other leukocytes, no DAPK1 or DAPK3 protein. When DAPK activities were blocked by inhibitors, we found that neither granulocyte lifespan nor phagocytosis was affected. However, such pharmacological inactivation of DAPK activity abolished motility of granulocytes in response to intermediary but not end-target chemoattractants ex vivo. The defect in chemotaxis in DAPK2-inactive granulocytes is likely a result of reduced polarization of the cells, mediated by a lack of MLC phosphorylation, resulting in radial F-actin and pseudopod formation. As neutrophils treated with DAPKi also showed reduced recruitment to the site of inflammation in a mouse peritonitis model, DAPK2 may be a novel target for anti-inflammatory therapies.

Dipeptidyl Peptidase IV Is a Human and Murine Neutrophil Chemorepellent

In Dictyostelium discoideum, AprA is a secreted protein that inhibits proliferation and causes chemorepulsion of Dictyostelium cells, yet AprA has little sequence similarity to any human proteins. We found that a predicted structure of AprA has similarity to human dipeptidyl peptidase IV (DPPIV). DPPIV is a serine protease present in extracellular fluids that cleaves peptides with a proline or alanine in the second position. In Insall chambers, DPPIV gradients below, similar to, and above the human serum DPPIV concentration cause movement of human neutrophils away from the higher concentration of DPPIV. A 1% DPPIV concentration difference between the front and back of the cell is sufficient to cause chemorepulsion. Neutrophil speed and viability are unaffected by DPPIV. DPPIV inhibitors block DPPIV-mediated chemorepulsion. In a murine model of acute respiratory distress syndrome, aspirated bleomycin induces a significant increase in the number of neutrophils in the lungs after 3 d. Oropharyngeal aspiration of DPPIV inhibits the bleomycin-induced accumulation of mouse neutrophils. These results indicate that DPPIV functions as a chemorepellent of human and mouse neutrophils, and they suggest new mechanisms to inhibit neutrophil accumulation in acute respiratory distress syndrome.

Localized bacterial infection induces systemic activation of neutrophils through Cxcr2 signaling in zebrafish

Neutrophils are the first line of defense against tissue damage and are rapidly mobilized to sites of bacterial infection. However, the signals that regulate neutrophil recruitment are not well defined. Here, using photolabel-enabled fate mapping in zebrafish larvae, we show that localized otic infection with Pseudomonas aeruginosa induces systemic activation and mobilization of neutrophils from the CHT through Cxcr2 signaling. We have cloned the zebrafish Cxcr1 and Cxcr2 receptors and show that Cxcr2 functions as a Cxcl8 receptor in live zebrafish. With the use of morpholino-mediated depletion, we show that infection-induced neutrophil mobilization from the CHT is mediated by Cxcr2 but not Cxcr1. By contrast, Cxcr2 depletion does not affect neutrophil recruitment to the chemoattractant LTB4. Taken together, our findings identify Cxcl8-Cxcr2 signaling as an infection-induced long-range cue that mediates neutrophil motility and mobilization from hematopoietic tissues, positioning Cxcr2 as a critical pathway that mediates infection-induced systemic activation of neutrophils.

Haematological features in Barth syndrome

This review highlights recent advances concerning pathogenesis, clinical presentation, diagnosis and treatment of Barth syndrome with particular regard to haematological abnormalities (e.g., neutropenia). Directed motility and killing activity of neutrophils is normal in patients with Barth syndrome, but neutrophils and eospinophils show phosphatidylserine exposure without exhibiting other markers of apoptosis. Apparently, neutropenia does not result from apoptosis of myeloid precursors or end-stage neutrophils but from reactive oxygen species triggered exposure of phosphatidylserine, leading to increased clearance of neutrophils by tissue macrophages. Lymphoblasts of patients with Barth syndrome show increased variability of mitochondrial size and increased mitochondrial mass due to increased clustering of fragmented mitochondria inside nuclear invaginations. Lymphoblast mitochondria show reduced cristae density, reduced cristae alignment and heterogenous cristae distribution. Areas of adhesion of opposing inner membranes result in obliteration of the inter-cristae space. Short or extended adhesion zones result in sheets of collapsed cristae, which are packaged as multiple concentric layers. In single patients, neutropenia may favourably respond to biweekly injections of granulocyte colony stimulating factor. Neutropenia in Barth syndrome is most likely due to reactive oxygen species induced exposure of phosphatidylserine, leading to increased clearance of neutrophils by tissue macrophages. In single patients, application of granulocyte colony stimulating factor may resolve neutropenia in Barth syndrome.

Hyaluronan Plus Saline Nasal Washes in the Treatment of Rhino-Sinusal Symptoms in Patients Undergoing Functional Endoscopic Sinus Surgery for Rhino-Sinusal Remodeling

Hyaluronic acid is a major component of many extracellular matrices and plays a central role in the homeostasis of physiology in upper and lower airways. When topically administered following endoscopic sinus surgery, hyaluronic acid may be effective in functional recovery and in the prevention of recurrence of chronic rhinosinusistis. This pilot study was aimed at evaluating the effects of nebulised 9 mg of sodium hyaluronate given for 15 days per months over 3 months in 46 patients aged greater than 4 years who underwent functional endoscopic sinus surgery (FESS) for rhino-sinusal remodelling. Eligible patients were randomized to receive nebulised 9 mg sodium hyaluronate nasal washes plus saline solution or 5 ml saline alone (23 patients in each group), according to an open-label, parallel group design, with blind observer assessment. Treatment was administered by means of a nasal ampoule that allows nebulisation of particles with a median aerodynamic diameter greater than 10 micron, i.e. suitable for upper respiratory airways deposition. The efficacy variables included clinical (presence of nasal dyspnoea), endoscopical (ostium of paranasal sinuses, oedema, respiratory patency, synechiae, and appearance of nasal mucosa) and cytological (ciliary motility and presence of neutrophils, eosinophils, mast cells, bacteria, mycetes and bio film) measures. At the end of the study, patients expressed an opinion on the overall tolerability of treatment. The two treatment groups were comparable at baseline. Treatment with 9 mg of sodium hyaluronate was associated with significantly greater improvements compared to controls in nasal dyspnoea (p less than 0.001), presence of mycetes (p = 0.044), ciliary motility (p less than 0.001) and abnormalities in nasal secretions. A univariate logistic model, in which the odd ratio (OR) indicates the probability of success in the 9 mg sodium hyaluronate group compared to the control group, showed that the highest OR was observed for presence of nasal dyspnoea (OR = 21.36; 95 percent CI: 1.07 to 426.56), normal mucosa at endoscopy (OR: 9.62; 95 percent CI: 1.82 to 50.89), ciliary motility (OR: 7.27; 95 percent CI: 1.68 to 31.42) and presence of bio film (OR: 4.41; 95 percent CI: 1.26 to 15.40). Treatment with 9 mg sodium hyaluronate plus saline was well tolerated. A 3-month intermittent treatment with 9 mg sodium hyaluronate plus saline solution nasal washes following FESS for rhino-sinusal remodelling was associated with significant improvements in nasal dyspnoea, appearance of nasal mucosa at endoscopy and ciliary motility compared to saline alone.

Phosphorylation of chemoattractant receptors regulates chemotaxis, actin re-organization, and signal-relay

SummaryMigratory cells, including mammalian leukocytes and Dictyostelium, use G-protein-coupled receptor (GPCR) signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are phosphorylated at cytoplasmic residues, uncoupling G-protein pathways, but activating other pathways. However, connections between GPCR phosphorylation and chemotaxis are unclear. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. cAMP oscillations derive from transient excitatory responses of adenylyl cyclase, which then rapidly adapts. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and show through chemotaxis modeling, single-cell FRET imaging, pure and chimeric population wavelet quantification, biochemical analyses and TIRF microscopy, that receptor phosphorylation is required to regulate adenylyl cyclase adaptation, long-range oscillatory cAMP wave production and cytoskeletal actin response. Phosphorylation defects thus promote hyperactive actin polymerization at the cell periphery, misdirected pseudopodia and the loss of directional chemotaxis. Our data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for migratory cell polarization and chemotaxis. Our results significantly extend the understanding of the function of GPCR phosphorylation, providing strong evidence that this evolutionarily conserved mechanism is required in a signal attenuation pathway that is necessary to maintain persistent directional movement of Dictyostelium, neutrophils and other migratory cells.

Fibronectin Induces Beta2-Integrin-Mediated Neutrophil Haptokinesis Independent of Chemoattractant

Neutrophils are mediators of innate immunity and inflammation and motility is critical to their function. The relationship between the presentation and identity of adhesive ligands and the dynamics of neutrophil motility is facilitated by the ability to manufacture well-defined adhesive surfaces that microcontact-printing affords. Here we show that by presenting fibronectin (FN) on poly(dimethylsiloxane) (PDMS) substrates, neutrophils are motile. This motility is haptokinetic and does not require prior or co-stimulation by chemoattractant. On printed fields of FN, the motility is phenotypically reminiscent of keratocytes and dependent upon the αMβ2 (Mac-1) integrin receptor. The phenotype observed contrasts with the classical hourglass morphology of neutrophils on FN adsorbed to glass. The addition of exogenous formylated chemoattractant increased the basal FN-induced motility in a dosage-dependent manner only when the FN concentration was below a critical threshold. These results suggest that neutrophils are capable of displaying multiple modes of motility as dictated by their adhesive environment. The exquisite cell-FN specificity demonstrated of neutrophils on printed PDMS makes the technique amenable to the future study of motility on more complex patterns of adhesive ligands as well as traction force mapping via micropost arrays.

Activation of AMPK Enhances Neutrophil Chemotaxis and Bacterial Killing

An inability of neutrophils to eliminate invading microorganisms is frequently associated with severe infection and may contribute to the high mortality rates associated with sepsis. In the present studies, we examined whether metformin and other 5' adenosine monophosphate-activated protein kinase (AMPK) activators affect neutrophil motility, phagocytosis and bacterial killing. We found that activation of AMPK enhanced neutrophil chemotaxis in vitro and in vivo, and also counteracted the inhibition of chemotaxis induced by exposure of neutrophils to lipopolysaccharide (LPS). In contrast, small interfering RNA (siRNA)-mediated knockdown of AMPKα1 or blockade of AMPK activation through treatment of neutrophils with the AMPK inhibitor compound C diminished neutrophil chemotaxis. In addition to their effects on chemotaxis, treatment of neutrophils with metformin or aminoimidazole carboxamide ribonucleotide (AICAR) improved phagocytosis and bacterial killing, including more efficient eradication of bacteria in a mouse model of peritonitis-induced sepsis. Immunocytochemistry showed that, in contrast to LPS, metformin or AICAR induced robust actin polymerization and distinct formation of neutrophil leading edges. Although LPS diminished AMPK phosphorylation, metformin or AICAR was able to partially decrease the effects of LPS/toll-like receptor 4 (TLR4) engagement on downstream signaling events, particularly LPS-induced IκBα degradation. The IκB kinase (IKK) inhibitor PS-1145 diminished IκBα degradation and also prevented LPS-induced inhibition of chemotaxis. These results suggest that AMPK activation with clinically approved agents, such as metformin, may facilitate bacterial eradication in sepsis and other inflammatory conditions associated with inhibition of neutrophil activation and chemotaxis.

Zebrafish Entrapment By Restriction Array (ZEBRA) device: a low-cost, agarose-free zebrafish mounting technique for automated imaging

The zebrafish has emerged as a useful model system for a variety of studies, including the investigation of inflammation and immunity. However, current zebrafish imaging techniques, such as agraose mounting, can be time-consuming and detrimental for long-term imaging. Alternatively, automated sorting and imaging systems can be costly and/or complicated to assemble. Here we describe the Zebrafish Entrapment by Restriction Array (ZEBRA) device, a microfluidic device that can be used to quickly and repeatably position zebrafish embryos in a predictable array using only a pipette. This technique is well suited for use with automated microscope stages leading to decreased imaging time and increased throughput compared to traditional methods. The addition of access ports above the embryo can be used to administer treatments, and potentially wounding or injections. We demonstrate the effectiveness of this device for a neutrophil migration screening application using larvae 3 days post fertilization (dpf) Tg(mpx:dendra2). Larvae were loaded into ZEBRA devices and treated with a neutrophil attractant (LTB4) or LTB4 with and without a PI3K inhibitor, LY294002. Treatment with LY294002 impaired neutrophil motility into the fin induced by LTB4 treatment. The findings report the development of ZEBRA a device that can be used to screen for small molecules that affect leukocyte motility and inflammation using live zebrafish.

Inflammatory Chemokines Direct and Restrict Leukocyte Migration within Live Tissues as Glycan-Bound Gradients

Chemokines are essential in many cell migration processes, including the recruitment of leukocytes to sites of infection. In the latter context, chemokines promote leukocyte extravasation into the relevant tissue through a well-studied cascade of events. It is widely believed that chemokines further guide leukocytes within tissues via chemotaxis, the directed migration along gradients of soluble ligands. However, the basic mechanism of chemokine action within tissues has yet to be formally addressed in vivo. We identified a chemokine (zCxcl8) that recruits zebrafish neutrophils to infection loci and analyzed its function directly within interstitial tissues of living larvae. Using noninvasive imaging and a controlled cellular source of zCxcl8, we found that zCxcl8 guides neutrophils in a 2-fold manner: by biasing cell speed according to direction (orthotaxis) and by restricting cell motility near the source. We further show that zCxcl8 establishes tissue-bound gradients in vivo by binding to heparan sulfate proteoglycans (HSPGs). Inhibition of this interaction compromised both directional guidance and restriction of neutrophil motility. Thus, by interacting with extracellular HSPGs, chemokines establish robust surface-bound (haptotactic) gradients that mediate both recruitment and retention of leukocytes at sites of infection.

Radil controls neutrophil adhesion and motility through β2-integrin activation

Integrin activation is required to facilitate multiple adhesion-dependent functions of neutrophils, such as chemotaxis, which is critical for inflammatory responses to injury and pathogens. However, little is known about the mechanisms that mediate integrin activation in neutrophils. We show that Radil, a novel Rap1 effector, regulates β1- and β2-integrin activation and controls neutrophil chemotaxis. On activation and chemotactic migration of neutrophils, Radil quickly translocates from the cytoplasm to the plasma membrane in a Rap1a-GTP-dependent manner. Cells overexpressing Radil show a substantial increase in cell adhesion, as well as in integrin/focal adhesion kinase (FAK) activation, and exhibit an elongated morphology, with severe tail retraction defects. This phenotype is effectively rescued by treatment with either β2-integrin inhibitory antibodies or FAK inhibitors. Conversely, knockdown of Radil causes severe inhibition of cell adhesion, β2-integrin activation, and chemotaxis. Furthermore, we found that inhibition of Rap activity by RapGAP coexpression inhibits Radil-mediated integrin and FAK activation, decreases cell adhesion, and abrogates the long-tail phenotype of Radil cells. Overall, these studies establish that Radil regulates neutrophil adhesion and motility by linking Rap1 to β2-integrin activation.

The Actin Regulatory Protein HS1 Interacts with Arp2/3 and Mediates Efficient Neutrophil Chemotaxis

HS1 is an actin regulatory protein and cortactin homolog that is expressed in hematopoietic cells. Antigen receptor stimulation induces HS1 phosphorylation, and HS1 is essential for T cell activation. HS1 is also expressed in neutrophils; however, the function of HS1 in neutrophils is not known. Here we show that HS1 localizes to the neutrophil leading edge, and is phosphorylated in response to the chemoattractant formyl-Met-Leu-Phe (fMLP) in adherent cells. Using live imaging in microchannels, we show that depletion of endogenous HS1 in the neutrophil-like PLB-985 cell line impairs chemotaxis. We also find that HS1 is necessary for chemoattractant-induced activation of Rac GTPase signaling and Vav1 phosphorylation, suggesting that HS1-mediated Rac activation is necessary for efficient neutrophil chemotaxis. We identify specific phosphorylation sites that mediate HS1-dependent neutrophil motility. Expression of HS1 Y378F, Y397F is sufficient to rescue migration of HS1-deficient neutrophils, however, a triple phospho-mutant Y222F, Y378F, Y397F did not rescue migration of HS1-deficient neutrophils. Moreover, HS1 phosphorylation on Y222, Y378, and Y397 regulates its interaction with Arp2/3. Collectively, our findings identify a novel role for HS1 and its phosphorylation during neutrophil directed migration.

Neutrophil Chemotaxis within a Competing Gradient of Chemoattractants

The dynamics of neutrophil chemotaxis under competing chemoattractant gradients was studied using a microfluidic platform. This microfluidic platform, which establishes a stable and dynamic gradient of chemoattractants across a cell culture chamber, enabled the investigation of human neutrophil migration patterns in the presences of four different chemoattractants (leukotriene B(4), chemokine C-X-C motif ligands 2 and 8, and fMLP) and competing gradients of all pairwise combinations. The migration patterns for individual cells were tracked and quantitatively analyzed, and the results suggest a hierarchy among these chemoattractants of fMLP > CXCL8 > CXCL2 > leukotriene B(4). In all conditions, over 60% of neutrophils exposed to a competing gradient move toward the stronger signal though the weaker chemoattractant still influences neutrophil motility. These results yield insight about how each chemoattractant contributes to overall neutrophil chemotaxis within complex physiological environments.

The UDP-sugar-sensing P2Y14 receptor promotes Rho-mediated signaling and chemotaxis in human neutrophils

The G(i)-coupled P2Y(14) receptor (P2Y(14)-R) is potently activated by UDP-sugars and UDP. Although P2Y(14)-R mRNA is prominently expressed in circulating neutrophils, the signaling pathways and functional responses associated with this receptor are undefined. In this study, we illustrate that incubation of isolated human neutrophils with UDP-glucose resulted in cytoskeleton rearrangement, change of cell shape, and enhanced cell migration. We also demonstrate that UDP-glucose promotes rapid, robust, and concentration-dependent activation of RhoA in these cells. Ecto-nucleotidases expressed on neutrophils rapidly hydrolyzed extracellular ATP, but incubation with UDP-glucose for up to 1 h resulted in negligible metabolism of the nucleotide-sugar. HL60 human promyelocytic leukemia cells do not express the P2Y(14)-R, but neutrophil differentiation of HL60 cells with DMSO resulted in markedly enhanced P2Y(14)-R expression. Accordingly, UDP-glucose, UDP-galactose, and UDP-N-acetylglucosamine promoted Rho activation in differentiated but not in undifferentiated HL60 cells. Stable expression of recombinant human P2Y(14)-R conferred UDP-sugar-promoted responses to undifferentiated HL60 cells. UDP-glucose-promoted RhoA activation also was accompanied by enhanced cell migration in differentiated HL60 cells, and these responses were blocked by Rho kinase inhibitors. These results support the notion that UDP-glucose is a stable and potent proinflammatory mediator that promotes P2Y(14)-R-mediated neutrophil motility via Rho/Rho kinase activation.

The Neutrophil's Eye-View: Inference and Visualisation of the Chemoattractant Field Driving Cell Chemotaxis In Vivo

As we begin to understand the signals that drive chemotaxis in vivo, it is becoming clear that there is a complex interplay of chemotactic factors, which changes over time as the inflammatory response evolves. New animal models such as transgenic lines of zebrafish, which are near transparent and where the neutrophils express a green fluorescent protein, have the potential to greatly increase our understanding of the chemotactic process under conditions of wounding and infection from video microscopy data. Measurement of the chemoattractants over space (and their evolution over time) is a key objective for understanding the signals driving neutrophil chemotaxis. However, it is not possible to measure and visualise the most important contributors to in vivo chemotaxis, and in fact the understanding of the main contributors at any particular time is incomplete. The key insight that we make in this investigation is that the neutrophils themselves are sensing the underlying field that is driving their action and we can use the observations of neutrophil movement to infer the hidden net chemoattractant field by use of a novel computational framework. We apply the methodology to multiple in vivo neutrophil recruitment data sets to demonstrate this new technique and find that the method provides consistent estimates of the chemoattractant field across the majority of experiments. The framework that we derive represents an important new methodology for cell biologists investigating the signalling processes driving cell chemotaxis, which we label the neutrophils eye-view of the chemoattractant field.