Neutrophil Motility Research Articles

A novel p38-MAPK signaling axis modulates neutrophil biology in head and neck cancer

Neutrophils are emerging as important mediators in cancer progression. Recent studies associated neutrophils with poor clinical outcome of HNC patients and showed that HNC induces recruitment, survival, and release of proinflammatory factors by neutrophils in vitro. The molecular mechanisms through which HNC and other cancers modulate neutrophil biology are currently unknown. To explore these mechanisms, we used an in vitro system that models the interaction between human HNC cells and neutrophils or neutrophilic-differentiated HL-60 cells, respectively. We show that HNC-derived factors activate p38-MAPK in neutrophils, which partly promotes neutrophil survival, but not neutrophil recruitment and motility. Most importantly, HNC-induced p38-MAPK activation strongly stimulates the release of CCL4, CXCL8, and MMP9 by neutrophils. We identify CREB and interestingly, p27 phosphorylated at T198 as downstream members of the HNC-induced p38-MAPK signaling cascade. Using siRNA technology, we demonstrate that p27 and CREB mediate the release of CCL4 and CXCL8 and that CREB, additionally, mediates the release of MMP9. These data unravel novel molecular mechanisms involved in regulation of neutrophil proinflammatory functions. Our studies on human HNC tissues indicate that tumor-infiltrating neutrophils might be a major source of CCL4 and particularly, MMP9 in cancer patients. Thus, our findings provide novel, mechanistic insights relevant for the pathophysiology of HNC and possibly, other types of cancer as well.

The SH2-domain-containing inositol 5-phosphatase (SHIP) limits neutrophil motility and wound recruitment in zebrafish

Neutrophil recruitment to sites of injury or infection is essential for host defense, but it needs to be tightly regulated to prevent tissue damage. Phosphoinositide 3-kinase (PI3K), which generates the phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P(3)], is necessary for neutrophil motility in vivo; however, the role of SH2-domain-containing 5-inositol phosphatase (SHIP) enzymes, which hydrolyze PI(3,4,5)P(3) to phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)], is not well understood. Here we show that SHIP phosphatases limit neutrophil motility in live zebrafish. Using real-time imaging of bioprobes specific for PI(3,4,5)P(3) and PI(3,4)P(2) in neutrophils, we found that PI(3,4,5)P(3) and PI(3,4)P(2) accumulate at the leading edge while PI(3,4)P(2) also localizes to the trailing edge of migrating neutrophils in vivo. Depletion of SHIP phosphatases using morpholino oligonucleotides led to increased neutrophil 3D motility and neutrophil infiltration into wounds. The increase in neutrophil wound recruitment in SHIP morphants was rescued by treatment with low dose PI3Kγ inhibitor, suggesting that SHIP limits neutrophil motility by modulating PI3K signaling. Moreover, overexpression of the SHIP phosphatase domain in neutrophils impaired neutrophil 3D migration. Taken together, our findings suggest that SHIP phosphatases control neutrophil inflammation by limiting neutrophil motility in vivo.

The role of microtubules in neutrophil polarity and migration in live zebrafish

Microtubules control cell motility by positively regulating polarization in many cell types. However, how microtubules regulate leukocyte migration is not well understood, particularly in living organisms. Here we exploited the zebrafish system to study the role of microtubules in neutrophil migration in vivo. The localization of microtubules was visualized in motile neutrophils using various bioprobes, revealing that, in contrast to what has been seen in studies in vitro, the microtubule organizing center is positioned in front of the nucleus (relative to the direction of migration) in motile neutrophils. Microtubule disassembly impaired attraction of neutrophils to wounds but enhanced the polarity of F-actin dynamics as measured by the distribution of stable and dynamic F-actin. Microtubule depolymerization inhibited polarized phosphoinositol 3-kinase (PI(3)K) activation at the leading edge and induced rapid PI(3)K independent motility. Finally, we show that microtubules exert their effects on neutrophil polarity and motility at least in part by the negative regulation of both Rho and Rac activity. These results provide new insight into the role of microtubules in neutrophil migration in a living vertebrate and show that the motility of these professional migratory cells are subject to distinctly different rules from those established for other cell types.

Bacillus anthracis-derived edema toxin (ET) counter-regulates movement of neutrophils and macromolecules through the endothelial paracellular pathway

BackgroundA common finding amongst patients with inhalational anthrax is a paucity of polymorphonuclear leukocytes (PMNs) in infected tissues in the face of abundant circulating PMNs. A major virulence determinant of anthrax is edema toxin (ET), which is formed by the combination of two proteins produced by the organism, edema factor (EF), which is an adenyl cyclase, and protective antigen (PA). Since cAMP, a product of adenyl cyclase, is known to enhance endothelial barrier integrity, we asked whether ET might decrease extravasation of PMNs into tissues through closure of the paracellular pathway through which PMNs traverse.ResultsPretreatment of human microvascular endothelial cell(EC)s of the lung (HMVEC-L) with ET decreased interleukin (IL)-8-driven transendothelial migration (TEM) of PMNs with a maximal reduction of nearly 60%. This effect required the presence of both EF and PA. Conversely, ET did not diminish PMN chemotaxis in an EC-free system. Pretreatment of subconfluent HMVEC-Ls decreased transendothelial 14 C-albumin flux by ~ 50% compared to medium controls. Coadministration of ET with either tumor necrosis factor-α or bacterial lipopolysaccharide, each at 100 ng/mL, attenuated the increase of transendothelial 14 C-albumin flux caused by either agent alone. The inhibitory effect of ET on TEM paralleled increases in protein kinase A (PKA) activity, but could not be blocked by inhibition of PKA with either H-89 or KT-5720. Finally, we were unable to replicate the ET effect with either forskolin or 3-isobutyl-1-methylxanthine, two agents known to increase cAMP.ConclusionsWe conclude that ET decreases IL-8-driven TEM of PMNs across HMVEC-L monolayers independent of cAMP/PKA activity.

Rho Family Proteins in High- and Low-Affinity fMLF Receptors Signaling in Neutrophils

Bacterial or mitochondrial peptide N-formyl-MLF (fMLF) binding with specific receptor activates chemotaxis, secretory degranulation and oxidative burst of the neutrophils. High and low affinity fMLF receptors (FPRs) can initiate different patterns of signaling components (Fu et al., 2004; Safronova, Gabdoulkhakova, 2009). Main functions of Rho-family proteins are signal transduction from various membrane receptors and regulation of the cytoskeleton. We suppose that small G-proteins are the node of FPRs signal transduction and divergence. Aim of the work was to find out the role of Rho family small G-proteins in signaling from high- and low-affinity receptors to NADPH oxidase. The study was carried out on the induced peritoneal neutrophils of Balb/c male mice. The immunofluorescent staining with specific Abs against RhoA and Rac1/2 was used to visualize the localization of Rho-proteins in the cells. Rho-proteins are evenly distributed in cytoplasm of non-stimulated cells. Translocation of Rho-GTPases to membrane occurs under the stimulation of neutrophils with fMLF that indicates their activation. High dose of fMLF (50 mkM) induces brighter fluorescence of juxtamembrane area in comparison with low dose (1 mkM). Formylated peptide fMLF stimulates dose-dependent activation of the neutrophil respiratory response. Rho activator reduces intensity of the respiratory burst under the stimulation of high-affinity receptors, but does not influence the respiratory burst initiated by low-affinity FPRs. Rac/Cdc42 activator also down-regulates the respiratory response to low concentration of fMLF without effect in case of high dose. Thus, Rho-family small GTPases participate in signal transduction from the FPRs and reduce activity of NADPH-oxidase.in the stimulation of the high-affinity FPRs. This effect of Rho-family proteins can endow the reciprocal relationships between chemotaxis and oxidative activity in process of the neutrophil movement within the gradient of the chemoattractants.

Actions of translocator protein ligands on neutrophil adhesion and motility induced by G-protein coupled receptor signaling

The 18kDa translocator protein (TSPO) also known as the peripheral benzodiazepine receptor (PBR), mediates the transportation of cholesterol and anions from the outer to the inner mitochondrial membrane in different cells types. Although recent evidences indicate a potential role for TSPO in the development of inflammatory processes, the mechanisms involved have not been elucidated. The present study investigated the ability of the specific TSPO ligands, the isoquinoline carboxamide PK11195 and benzodiazepine Ro5-4864, on neutrophil recruitment promoted by the N-formylmethionyl-leucyl-phenylalanine peptide (fMLP), an agonist of G-protein coupled receptor (GPCR). Pre-treatment with Ro5-4864 abrograted fMLP-induced leukocyte-endothelial interactions in mesenteric postcapillary venules in vivo. Moreover, in vitro Ro5-4864 treatment prevented fMLP-induced: (i) l-selectin shedding and overexpression of PECAM-1 on the neutrophil cell surface; (ii) neutrophil chemotaxis and (iii) enhancement of intracellular calcium cations (iCa+2). Intriguingly, the two latter effects were augmented by cell treatment with PK11195. An allosteric agonist/antagonist relation may be suggested, as the effects of Ro5-4864 on fMLP-stimulated neutrophils were reverted by simultaneous treatment with PK11195. Taken together, these data highlight TSPO as a modulator of pathways of neutrophil adhesion and locomotion induced by GPCR, connecting TSPO actions and the onset of an innate inflammatory response.

The Janus Face of Neutrophil Specific Antigen CD177 in Bacterial Infection: Focal Disease Versus Systemic Disease

Abstract 1108CD177, also known as NB1 (or HNA-2) is a GPI-linked glycoprotein, which is exclusively expressed on neutrophils. Approximately 3 to 5% of healthy individuals do not express this antigen on their neutrophils (NBnull).Recent data demonstrated a strong upregulation of NB1 on neutrophils in patients with bacterial, but not viral infections. The mechanism underlying this phenomenon, however, is unknown. Our previous studies showed that NB1 functions as a partner of endothelial PECAM-1 and therefore plays a role on neutrophil diapedesis. Consequently, neutrophils carrying NB1 (NB1plus) migrate faster through endothelial cells than NBnull neutrophils. However, several studies have documented an abrogation of neutrophil migratory abilities in sepsis conditions. In this study, we aim to clarify the impact of neutrophil NB1 expression in bacterial sepsis.To mimic this condition in vitro, we first compared the transendothelial migration ability of NB1 phenotyped neutrophils after stimulation with the bacterial peptide fMLP in a transwell system. Lower transmigration ability (75% vs. 40%) was observed in fMLP-treated NB1plus neutrophils (n = 5) in comparison to untreated neutrophils. In contrast, no significant difference in the migration ability was observed between fMLP-treated and untreated NB1null neutrophils (n = 3). Expression analysis by flow cytometry showed a dose-dependent upregulation of NB1 after stimulation with fMLP (10−6 to 10−8 μM) in NB1plus neutrophils. Interestingly, down regulation of PECAM-1 expression was observed in these treated cells. Contrary, no PECAM-1 downregulation was detected in NBnull fMLP-treated neutrophils. These results could be confirmed by immunoblotting analysis using specific antibodies directed against different epitopes on NB1 (mabs 7D8, MEM166) and against different PECAM-1 Ig domains (mabs PECAM1.1, 1.2 and 1.3). Analysis of the supernatants of fMLP-treated neutrophils demonstrated the shedding of PECAM-1 from NB1plus, but not from NB1null neutrophils. These results indicate that shedding of PECAM-1 from neutrophils during bacterial infections depends on NB1 expression.Recent studies showed that a single nucleotide polymorphism (42C>G) located in NB1 promoter region is associated with the regulation of NB1 expression. Individuals homozygous for C allele express high NB1 surface density in comparison to individuals homozygous for G allele. Our association study showed higher frequency of C allele in patients with bacterial sepsis (n=98) compared with healthy cohort (n = 132) (8.16% vs. 12.88%; P<0.03). This observation indicates the role of C42 allele (or high NB1 expression) as genetic risk factor for bacterial sepsis.All together, these data demonstrate a reverse role of NB1 expression in focal and systemic infection, indicating favourable effect of low NB1 in systemic bacterial infection. This phenomenon may be caused by reduction of neutrophil directionality and motility due to NB1-mediated PECAM-1 shedding. Disclosures:No relevant conflicts of interest to declare.

Acute pyelonephritis and renal scarring are caused by dysfunctional innate immunity in mCxcr2 heterozygous mice

The CXCR1 receptor and chemokine CXCL8 (IL-8) support neutrophil-dependent clearance of uropathogenic Escherichia coli from the urinary tract. CXCR1 is reduced in children prone to pyelonephritis, and heterozygous hCXCR1 polymorphisms are more common in this patient group than in healthy individuals, strongly suggesting a disease association. Since murine CXCR2 (mCXCR2) is functionally similar to human CXCR1, we determined effects of gene heterozygosity on the susceptibility to urinary tract infection by infecting heterozygous (mCxcr2+/−) mice with uropathogenic Escherichia coli. Clearance of infection and tissue damage were assessed as a function of innate immunity in comparison to that in knockout (mCxcr2−/−) and wild-type (mCxcr2+/+) mice. Acute sepsis-associated mortality was increased and bacterial clearance drastically impaired in heterozygous compared to wild-type mice. Chemokine and neutrophil responses were delayed along with evidence of neutrophil retention and unresolved kidney inflammation 1 month after infection. This was accompanied by epithelial proliferation and subepithelial fibrosis. The heterozygous phenotype was intermediate, between knockout and wild-type mice, but specific immune cell infiltrates that accompany chronic infection in knockout mice were not found. Hence, the known heterozygous CXCR1 polymorphisms may predispose patients to acute pyelonephritis and urosepsis.

Dual Roles for Rac2 in Neutrophil Motility and Active Retention in Zebrafish Hematopoietic Tissue

Neutrophil homeostasis is essential for host defense. Here we identify dual roles for Rac2 during neutrophil homeostasis using a zebrafish model of primary immune deficiency induced by the human inhibitory Rac2D57N mutation in neutrophils. Noninvasive live imaging of Rac2 morphants or Rac2D57N zebrafish larvae demonstrates an essential role for Rac2 in regulating 3D motility and the polarization of F-actin dynamics and PI(3)K signaling invivo. Tracking of photolabeled Rac2-deficient neutrophils from hematopoietic tissue also shows increased mobilization into the circulation, indicating that neutrophil mobilization does not require traditionally defined cell motility. Moreover, excessive neutrophil retention in hematopoietic tissue resulting from a constitutively active CXCR4 mutation in zebrafish warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is partially rescued by the inhibitory Rac2 mutation. These findings reveal that Rac2 signaling is necessary for both neutrophil 3D motility and CXCR4-mediated neutrophil retention in hematopoietic tissue, thereby limiting neutrophil mobilization, a critical first step in the innate immune response.

Silencer of Death Domains (SODD) Inhibits Skeletal Muscle and Kidney Enriched Inositol 5-Phosphatase (SKIP) and Regulates Phosphoinositide 3-Kinase (PI3K)/Akt Signaling to the Actin Cytoskeleton

Phosphoinositide 3-kinase (PI3K) regulates cell polarity and migration by generating phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) at the leading edge of migrating cells. The serine-threonine protein kinase Akt binds to PI(3,4,5)P(3), resulting in its activation. Active Akt promotes spatially regulated actin cytoskeletal remodeling and thereby directed cell migration. The inositol polyphosphate 5-phosphatases (5-ptases) degrade PI(3,4,5)P(3) to form PI(3,4)P(2), which leads to diminished Akt activation. Several 5-ptases, including SKIP and SHIP2, inhibit actin cytoskeletal reorganization by opposing PI3K/Akt signaling. In this current study, we identify a molecular co-chaperone termed silencer of death domains (SODD/BAG4) that forms a complex with several 5-ptase family members, including SKIP, SHIP1, and SHIP2. The interaction between SODD and SKIP exerts an inhibitory effect on SKIP PI(3,4,5)P(3) 5-ptase catalytic activity and consequently enhances the recruitment of PI(3,4,5)P(3)-effectors to the plasma membrane. In contrast, SODD(-/-) mouse embryonic fibroblasts exhibit reduced Akt-Ser(473) and -Thr(308) phosphorylation following EGF stimulation, associated with increased SKIP PI(3,4,5)P(3)-5-ptase activity. SODD(-/-) mouse embryonic fibroblasts exhibit decreased EGF-stimulated F-actin stress fibers, lamellipodia, and focal adhesion complexity, a phenotype that is rescued by the expression of constitutively active Akt1. Furthermore, reduced cell migration was observed in SODD(-/-) macrophages, which express the three 5-ptases shown to interact with SODD (SKIP, SHIP1, and SHIP2). Therefore, this study identifies SODD as a novel regulator of PI3K/Akt signaling to the actin cytoskeleton.

Hax1 regulates neutrophil adhesion and motility through RhoA

Hax1 regulates neutrophil adhesion and motility through RhoA

Hax1 regulates neutrophil adhesion and motility through RhoA

Kostmann disease is an inherited severe congenital neutropenia syndrome associated with loss-of-function mutations in an adaptor protein HS1-associated protein X-1 (Hax1). How Hax1 regulates neutrophil function remains largely unknown. In this paper, we use ribonucleic acid interference to deplete Hax1 in the neutrophil-like cell line PLB-985 and identify Hax1 as a negative regulator of integrin-mediated adhesion and chemotaxis. Using microfluidics, we show that depletion of Hax1 impairs neutrophil uropod detachment and directed migration. Hax1-deficient cells also display increased integrin-mediated adhesion and reduced RhoA activity. Moreover, depletion of RhoA induces increased neutrophil adhesion and impaired migration, suggesting that Hax1 regulates neutrophil adhesion and chemotaxis through RhoA. Accordingly, activation of RhoA is sufficient to rescue adhesion of Hax1-deficient neutrophils. Together, our findings identify Hax1 as a novel regulator of neutrophil uropod detachment and chemotaxis through RhoA.

G-CSF–mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands

AbstractEmergency mobilization of neutrophil granulocytes (neutrophils) from the bone marrow (BM) is a key event of early cellular immunity. The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) stimulates this process, but it is unknown how individual neutrophils respond in situ. We show by intravital 2-photon microscopy that a systemic dose of human clinical-grade G-CSF rapidly induces the motility and entry of neutrophils into blood vessels within the tibial BM of mice. Simultaneously, the neutrophil-attracting chemokine KC (Cxcl1) spikes in the blood. In mice lacking the KC receptor Cxcr2, G-CSF fails to mobilize neutrophils and antibody blockade of Cxcr2 inhibits the mobilization and induction of neutrophil motility in the BM. KC is expressed by megakaryocytes and endothelial cells in situ and is released in vitro by megakaryocytes isolated directly from BM. This production of KC is strongly increased by thrombopoietin (TPO). Systemic G-CSF rapidly induces the increased production of TPO in BM. Accordingly, a single injection of TPO mobilizes neutrophils with kinetics similar to G-CSF, and mice lacking the TPO receptor show impaired neutrophil mobilization after short-term G-CSF administration. Thus, a network of signaling molecules, chemokines, and cells controls neutrophil release from the BM, and their mobilization involves rapidly induced Cxcr2-mediated motility controlled by TPO as a pacemaker.

Guided by the Light: Neutrophil Migration through Zebrafish

second sentence in the first issue of Developmental Cell states that the new journal's goal is to provide a unique resource for scientists interested in the properties of individual cells and their regulation in a larger multicellular context. paper by Yoo, Huttenlocher, and colleagues illustrates this credo beautifully. Using live imaging, the authors analyzed how individual neutrophils navigate through the zebrafish embryo. Most strikingly, local activation of a photoactivatable form of Rac induced membrane protrusions and directed migration. PI(3)K inhibition abolished light-guided migration, suggesting that PI(3)K does not act exclusively through Rac. paper not only provides an elegant test of the roles of Rac and PI(3)K during migration but also highlights the power of optogenetic technologies in analyzing and manipulating cells during development. Or, as stated in the very first issue, The field is flourishing, and new tools are opening up avenues of investigation that would have seemed daunting if not unattainable a few years ago. This PaperPick refers to Differential Regulation of Protrusion and Polarity by PI(3)K during Neutrophil Motility in Live Zebrafish by Sa Kan Yoo, Qing Deng, Peter J. Cavnar, Yi I. Wu, Klaus M. Hahn, and Anna Huttenlocher, published in February, 2010. Video Abstract Anna Huttenlocher, with the help of Sa Kan Yoo and Qing Deng, explain their investigation of neutrophil migration through the zebrafish embryo.

Modulation of neutrophil motility by curcumin

Neutrophils (PMN) are the first cells recruited at the site of inflammation. They play a key role in the innate immune response by recognizing, ingesting, and eliminating pathogens and participate in the orientation of the adaptive immune responses. However, in inflammatory bowel disease (IBD) transepithelial neutrophil migration leads to an impaired epithelial barrier function, perpetuation of inflammation, and tissue destruction via oxidative and proteolytic damage. Curcumin (diferulolylmethane) displays a protective role in mouse models of IBD and in human ulcerative colitis, a phenomenon consistently accompanied by a reduced mucosal neutrophil infiltration. We investigated the effect of curcumin on mouse and human neutrophil polarization and motility in vitro and in vivo. Curcumin attenuated lipopolysaccharide (LPS)-stimulated expression and secretion of macrophage inflammatory protein (MIP)-2, interleukin (IL)-1β, keratinocyte chemoattractant (KC), and MIP-1α in colonic epithelial cells (CECs) and in macrophages. Curcumin significantly inhibited PMN chemotaxis against MIP-2, KC, or against conditioned media from LPS-treated macrophages or CEC, a well as the IL-8-mediated chemotaxis of human neutrophils. At nontoxic concentrations, curcumin inhibited random neutrophil migration, suggesting a direct effect on neutrophil chemokinesis. Curcumin-mediated inhibition of PMN motility could be attributed to a downregulation of PI3K activity, AKT phosphorylation, and F-actin polymerization at the leading edge. The inhibitory effect of curcumin on neutrophil motility was further demonstrated in vivo in a model of aseptic peritonitis. Our results indicate that curcumin interferes with colonic inflammation partly through inhibition of the chemokine expression and through direct inhibition of neutrophil chemotaxis and chemokinesis.

Maltooligosaccharides from JEG-3 Trophoblast-Like Cells Exhibit Immunoregulatory Properties

to better understand the immunoregulatory properties of trophoblasts, we have searched for small immunologically active carbohydrates derived from intact trophoblast-like cells. using solid phase extraction coupled with HPLC and mass spectrometry methods, we have characterized a low molecular weight carbohydrate-rich fraction associated with JEG-3 cells. We have also tested the bioactivities of selected authentic oligosaccharides found in the oligosaccharide fraction. the most abundant components of the low molecular weight carbohydrate-rich fraction were maltotriose and maltotetraose, with detectable amounts of maltopentaose. When authentic maltooligosaccharides were tested using lymphocytes, IL-2 inhibition was observed. This activity was dependent upon the number of saccharide subunits, stereochemistry, and concentration. To further test maltooligosaccharide properties, maltopentose was attached to glass cover slips. Although spontaneous neutrophil motility was observed on unmodified and control surfaces, it was inhibited on maltooligosaccharide-derivatized surfaces. maltooligosaccharides are associated with the trophoblast's surface where they may exhibit immunoregulatory activities.

Live imaging of neutrophil motility in a zebrafish model of WHIM syndrome

AbstractCXCR4 is a G protein–coupled chemokine receptor that has been implicated in the pathogenesis of primary immunodeficiency disorders and cancer. Autosomal dominant gain-of-function truncations of CXCR4 are associated with warts, hypo-gammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a primary immunodeficiency disorder characterized by neutropenia and recurrent infections. Recent progress has implicated CXCR4-SDF1 (stromal cell-derived factor 1) signaling in regulating neutrophil homeostasis, but the precise role of CXCR4-SDF1 interactions in regulating neutrophil motility in vivo is not known. Here, we use the optical transparency of zebrafish to visualize neutrophil trafficking in vivo in a zebrafish model of WHIM syndrome. We demonstrate that expression of WHIM mutations in zebrafish neutrophils induces neutrophil retention in hematopoietic tissue, impairing neutrophil motility and wound recruitment. The neutrophil retention signal induced by WHIM truncation mutations is SDF1 dependent, because depletion of SDF1 with the use of morpholino oligonucleotides restores neutrophil chemotaxis to wounds. Moreover, localized activation of a genetically encoded, photoactivatable Rac guanosine triphosphatase is sufficient to direct migration of neutrophils that express the WHIM mutation. The findings suggest that this transgenic zebrafish model of WHIM syndrome may provide a valuable tool to screen for agents that modify CXCR4-SDF1 retention signals.

Direction of neutrophil movements by Campylobacter-infected intestinal epithelium

Campylobacter jejuni and Campylobacter coli together represent the leading bacterial cause of human enteritis. However, the pathogenesis of this disease is poorly understood. Infection results in the formation of crypt abscesses resulting from the migration of neutrophils across the intestinal epithelium and into the intestinal crypts. In this study, we model this process in vitro and show that Campylobacter infection of epithelium results in a quantifiable increase in the directed movement of neutrophils from the basolateral to apical surface of the epithelium. This process is dependent on both bacterially derived n-formyl peptides and on the host cell enzyme 12-lipoxygenase (12-LOX).

Leukocyte Chemotaxis and Migration

Leukocyte Chemotaxis and Migration

Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices

Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72–120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.